U.S. patent application number 16/277721 was filed with the patent office on 2019-08-22 for protein modification of living cells using sortase.
This patent application is currently assigned to Whitehead Institute for Biomedical Research. The applicant listed for this patent is Whitehead Institute for Biomedical Research. Invention is credited to Hidde L. Ploegh, Lee Kim Swee.
Application Number | 20190256818 16/277721 |
Document ID | / |
Family ID | 51867884 |
Filed Date | 2019-08-22 |
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United States Patent
Application |
20190256818 |
Kind Code |
A1 |
Swee; Lee Kim ; et
al. |
August 22, 2019 |
PROTEIN MODIFICATION OF LIVING CELLS USING SORTASE
Abstract
Non-genetically engineered mammalian cells modified by
sortase-mediated conjugation of an agent thereto are provided.
Methods of conjugating agents to non-genetically engineered
mammalian cells using sortase are provided. Methods of using the
cells, e.g., for diagnostic and/or therapeutic purposes, are
provided.
Inventors: |
Swee; Lee Kim; (Heidelberg,
DE) ; Ploegh; Hidde L.; (Jamaica Plain, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Whitehead Institute for Biomedical Research |
Cambridge |
MA |
US |
|
|
Assignee: |
Whitehead Institute for Biomedical
Research
Cambridge
MA
|
Family ID: |
51867884 |
Appl. No.: |
16/277721 |
Filed: |
February 15, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14890296 |
Nov 10, 2015 |
10260038 |
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PCT/US2014/037545 |
May 9, 2014 |
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16277721 |
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61943094 |
Feb 21, 2014 |
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61822092 |
May 10, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2039/5158 20130101;
C12N 9/50 20130101; C12N 9/52 20130101; A61P 35/02 20180101; C12N
5/0006 20130101; A61P 31/10 20180101; A61K 47/65 20170801; A61P
31/12 20180101; A61P 31/04 20180101; C12N 5/0638 20130101; A61K
47/66 20170801; C12Y 304/2207 20130101; A61K 39/0011 20130101; A61P
35/00 20180101; A61P 43/00 20180101; C12N 5/0636 20130101; A61P
33/00 20180101; A61K 47/68 20170801 |
International
Class: |
C12N 5/00 20060101
C12N005/00; A61K 47/68 20060101 A61K047/68; C12N 5/0783 20060101
C12N005/0783; A61K 47/65 20060101 A61K047/65; C12N 9/50 20060101
C12N009/50; A61K 47/66 20060101 A61K047/66; A61K 39/00 20060101
A61K039/00 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] The invention was made with government support under Grant
No. R01 A1087879 awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
1-141. (canceled)
142. A human cell having an agent linked thereto via a sortase
recognition sequence, wherein the agent is linked to an endogenous,
non-genetically engineered protein of the human cell, and wherein
the human cell has not been genetically engineered to express a
protein comprising a sortase recognition sequence, and wherein the
agent comprises a cytokine, an antigen, a costimulatory molecule,
or an adjuvant.
143. The human cell of claim 142, wherein the human cell is a
cellular artificial antigen presenting cell (aAPC).
144. The human cell of claim 142, wherein the agent comprises an
antigen, and wherein the antigen is a tumor antigen, a viral
antigen, a bacterial antigen, a fungal antigen, or a parasite
antigen.
145. The human cell of claim 142, wherein the human cell is a
hematopoietic stem cell (HSC).
146. The human cell of claim 142, wherein the human cell is a
myeloid progenitor cell or a lymphoid progenitor cell.
147. The human cell of claim 142, wherein the human cell is a red
blood cell.
148. The human cell of claim 142, wherein the human cell is
selected from the group consisting of a lymphocyte, a monocyte, a
dendritic cell, a macrophage, a neutrophil, a mast cell, an
eosinophil, a basophil, and a natural killer (NK) cell.
149. The human cell of claim 142, wherein the costimulatory
molecule is a molecule that binds to a CD28 family receptor, a CD2
family receptor, ICOS, CD27, or 4-1BBL.
150. The human cell of claim 142, wherein the agent comprises a
costimulatory molecule, and the costimulatory molecule is a B7
molecule, an ICOS ligand, a TNF alpha family member 4-1BBL, OX40,
or OX40L.
151. The human cell of claim 142, wherein the agent comprises a
costimulatory molecule, and the costimulatory molecule is
4-1BBL.
152. The human cell of claim 142, wherein the agent comprises an
adjuvant, and wherein the adjuvant comprises a CD40 ligand,
anti-CD40 antibody, a ligand of a TLR, a pathogen-derived molecular
pattern (PAMP), a PAMP mimic, an immunostimulatory nucleic acid, or
a cationic polymer.
153. The human cell of claim 152, wherein the adjuvant comprises a
ligand of a TLR, and wherein the TLR is TLR3, TLR4, and/or
TLR9.
154. The human cell of claim 152, wherein the adjuvant comprises a
cationic polymer, and wherein the cationic polymer is a poly(amino
acid).
155. The human cell of claim 142, wherein the agent comprises a
cytokine, and wherein the cytokine is an interleukin, TNF-alpha, a
colony stimulating factor, interferon alpha 2a, interferon alpha
2b, interferon beta-1a, interferon beta-1b, leukemia inhibitory
factor (LIF), or oncostatin M.
156. The human cell of claim 155, wherein the cytokine is an
interleukin, and wherein the interleukin is IL-1, IL-2, IL-3, IL-4,
IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14,
membrane-bound IL-15, IL-15, IL-17, IL-21, IL-23, IL-27, IL-35, or
IL-38.
157. The human cell of claim 155, wherein the cytokine is an
interleukin, and wherein the interleukin is IL-15 or membrane-bound
IL-15.
158. The human cell of claim 155, wherein the cytokine is a colony
stimulating factor, and wherein the colony stimulating factor is
granulocyte colony-stimulating factor (G-CSF), granulocyte
macrophage colony-stimulating factor (GM-CSF), or macrophage
colony-stimulating factor (M-CSF).
159. The human cell of claim 142, wherein the sortase recognition
sequence comprises LPXTG.
160. A method of modulating an immune response of a subject to an
entity of interest, the method comprising administering to the
subject the human cell of claim 142.
161. A method of treating a subject in need of treatment for a
disease, the method comprising administering to the subject the
human cell of claim 142.
162. The method of claim 161, wherein the disease comprises an
infectious disease, cancer, an autoimmune disease, an allergy, an
inflammatory condition, or an immunodeficiency.
163. A method of conjugating an agent to a human cell, the method
comprising contacting the human cell with a sortase substrate that
comprises both a sortase recognition sequence and an agent, wherein
the contacting is performed in the presence of a sortase under
conditions suitable for the sortase to conjugate the sortase
substrate to an endogenous, non-engineered polypeptide of the human
cell, wherein the agent comprises a cytokine, an antigen, a
costimulatory molecule, or an adjuvant.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of and claims priority
under 35 U.S.C. .sctn. 120 to U.S. patent application U.S. Ser. No.
14/890,296, filed Nov. 10, 2015, which is a national phase filing
under 35 U.S.C. .sctn. 371 of international PCT application,
PCT/US2014/037545, filed May 9, 2014, which claims priority under
35 U.S.C. .sctn. 119(e) to U.S. Provisional Application, U.S. Ser.
No. 61/822,092, filed on May 10, 2013, and to U.S. Provisional
Application, U.S. Ser. No. 61/943,094, filed on Feb. 21, 2014, each
of which is incorporated herein by reference.
BACKGROUND
[0003] Bacterial sortases were originally identified as enzymes
that covalently attach proteins to the bacterial cell wall. For
example, Staphylococcus aureus sortase A recognizes a set of
diverse substrates via a sortase recognition motif (e.g., LPXTG)
and cleaves the peptide bond between threonine and glycine, thereby
releasing the residues C-terminal to the threonine and yielding an
amide linkage with the N terminus of a pentaglycine nucleophile,
which is provided in vivo by a cell wall precursor.
[0004] The transpeptidation reaction catalyzed by sortases has
emerged as a versatile method for site-specific modification of
proteins and has been applied to a variety of in vitro reactions.
The method has proved versatile in part because the enzyme
tolerates a wide variety of substrates in proximity of the cleavage
site and in nucleophiles. In many sortase-based protein
modification methods a protein to be modified is engineered to
contain a sortase recognition motif (e.g., LPXTG) at or near its
C-terminus. When incubated with sortase and a synthetic peptide
containing one or more N-terminal glycine residues, such artificial
sortase substrates undergo a transacylation reaction resulting in
the exchange of residues C-terminal to the threonine residue with
the synthetic peptide, resulting in the protein C-terminus being
ligated to the N-terminus of the synthetic peptide. In some cases,
a protein to be modified is engineered to contain one or more
N-terminal glycine residues near its N-terminus. When incubated
with sortase and a synthetic peptide containing a sortase
recognition motif and a sortase, the transacylation reaction
results in the exchange of residues C-terminal to the threonine
residue in the synthetic peptide with the modified protein,
resulting in the synthetic peptide being ligated to the N-terminus
of the protein. The synthetic peptides used in either approach may
be fused or conjugated to any of a number of different moieties.
When the synthetic peptide and protein are conjugated via
sortase-mediated transacylation, such moieties become attached to
the protein.
[0005] The sortase-catalyzed reaction has been used for, among
other things, ligating proteins and/or peptides to one another in
vitro, conjugating a protein or peptide to a solid support or
polymer, and linking a label to a protein or peptide.
SUMMARY
[0006] Some aspects of this invention relate to sortase-mediated
modification of proteins expressed by living animal cells, wherein
the cells are not genetically engineered to express a protein
comprising a sortase recognition sequence or a sequence capable of
serving as a nucleophilic acceptor sequence in a sortase-mediated
reaction. In some embodiments the animal cells are not genetically
engineered. In some embodiments the animal cells are mammalian
cells, e.g., human cells. In some embodiments the cells are immune
system cells. In some embodiments the methods provide for attaching
any moiety of interest to a living animal cell, without requiring
that the animal cell be genetically engineered and without
requiring the use of crosslinking reagents.
[0007] Any of a wide variety of agents may be conjugated to a
protein expressed by an animal cell in accordance with various
embodiments. In some embodiments, a protein is modified by the
conjugation of a sortase substrate comprising an amino acid, a
peptide, a protein, a polynucleotide, a carbohydrate, a tag, a
metal atom, a contrast agent, a catalyst, a non-polypeptide
polymer, a recognition element, a small molecule, a lipid, a
linker, a label, an epitope, an antigen, a therapeutic agent, a
toxin, a radioisotope, a particle, or moiety comprising a reactive
chemical group, e.g., a click chemistry handle.
[0008] In some embodiments, a method comprises contacting a living
animal cell with a sortase and a sortase substrate comprising a
sortase recognition motif, wherein the animal cell is not
genetically engineered to express a protein comprising a sortase
recognition sequence or a sequence capable of serving as a
nucleophilic acceptor sequence in a reaction catalyzed by the
sortase. In some embodiments the animal cell is not genetically
engineered. In some embodiments contacting is performed under
conditions suitable for the sortase to transamidate the sortase
substrate and a polypeptide exposed at the surface of the animal
cell, thereby conjugating the sortase substrate to the polypeptide.
In some embodiments the sortase substrate comprises a sortase
recognition motif and a moiety of interest, e.g., an amino acid, a
peptide, a protein, a polynucleotide, a carbohydrate, a tag, a
metal atom, a contrast agent, a catalyst, a non-polypeptide
polymer, a recognition element, a small molecule, a lipid, a
linker, a label, an epitope, an antigen, a therapeutic agent, a
toxin, a radioisotope, a particle, or a click chemistry handle.
Conjugating the sortase substrate to the polypeptide exposed at the
surface of animal cell attaches the moiety of interest to the cell
expressing the polypeptide.
[0009] In some embodiments a sortase substrate comprises an
antibody, e.g., a single chain antibody such as a camelid antibody,
a single-domain antibody, a VHH domain, a nanobody, or an scFv. In
some embodiments a sortase substrate comprises a binding moiety. In
some embodiments a binding moiety may comprise an antibody,
polypeptide, affibody, adnectin, anticalin, or aptamer. In some
embodiments a binding moiety may serve as a targeting moiety. In
some embodiments a binding moiety binds to a cell surface marker of
a target cell. In some embodiments a target cell is a cancer cell,
infected cell, or other abnormal or diseased cell. In some
embodiments a target cell is a normal cell.
[0010] In some embodiments a sortase substrate comprises a click
chemistry handle. Click chemistry handles are chemical moieties
that provide a reactive group that can partake in a click chemistry
reaction. Click chemistry reactions and suitable chemical groups
for click chemistry reactions are well known to those of skill in
the art, and include, but are not limited to terminal alkynes,
azides, strained alkynes, dienes, dieneophiles, alkoxyamines,
carbonyls, phosphines, hydrazides, thiols, and alkenes. For
example, in some embodiments, an azide and an alkyne are used in a
click chemistry reaction. In some embodiments a reactive group of
first click chemistry handle attached to an animal cell via a
sortase-catalyzed reaction is reacted with a second reactive group
attached to a second entity, thereby conjugating the second entity
to the animal cell. The second reactive group may be a second click
chemistry handle that is compatible with the first click chemistry
handle. The entity may be, e.g., an amino acid, a peptide, a
protein, a polynucleotide, a carbohydrate, a tag, a metal atom, a
contrast agent, a catalyst, a non-polypeptide polymer, a
recognition element, a small molecule, a lipid, a linker, a label,
an epitope, an antigen, a therapeutic agent, a toxin, a
radioisotope, a particle, or a cell. In some embodiments the entity
may be an antibody, e.g., a single chain antibody such as a camelid
antibody, a single-domain antibody, a VHH domain, a nanobody, or an
scFv. In some embodiments the entity comprises a binding moiety. In
some embodiments a binding moiety may comprise an antibody,
polypeptide, affibody, adnectin, anticalin, or aptamer.
[0011] Some aspects of this invention provide animal cells
comprising one or more modified endogenous, non-genetically
engineered proteins comprising an agent conjugated at or near its
N-terminus. In some embodiments the agent comprises a moiety of
interest, e.g., an amino acid, a peptide, a protein, a
polynucleotide, a carbohydrate, a tag, a metal atom, a contrast
agent, a catalyst, a non-polypeptide polymer, a recognition
element, a small molecule, a lipid, a linker, a label, an epitope,
an antigen, a therapeutic agent, a toxin, a radioisotope, a
particle, or a click chemistry handle. In some embodiments, the
modified endogenous protein comprises an antigen-binding domain,
for example, an antigen-binding domain of an antibody, e.g., a
camelid antibody, a single-domain antibody, a VHH domain, a
nanobody, or an ScFv.
[0012] Some aspects of this invention comprise administering
modified modified mammalian cells, e.g., modified human cells, to
subjects, e.g., human subjects. In some embodiments the modified
mammalian cells enhance the subject's immune response to a cancer
cell, infected cell, or other abnormal cell, or directly attack a
cancer cell, infected cell, or other abnormal cell. In some
embodiments the modified mammalian cells have a therapeutic agent
or detection agent conjugated to an endogenous protein and serve to
deliver the agent to the subject.
[0013] Some embodiments of this invention provide chimeric
proteins, for example, chimeric proteins that have been generated
by conjugation of two proteins, wherein at least one of the
proteins is an endogenous protein expressed by a living animal
cell. Some embodiments provide living animal cells, e.g., mammalian
cells, having one or more such chimeric proteins attached to their
surface.
[0014] Some embodiments provide modified modified endogenous
mammalian proteins comprising a sortase recognition motif (e.g.,
LPXTG) and a moiety attached to the sortase recognition motif. For
example, a moiety may be attached directly to one of the amino
acids of the sortase recognition motif or may be attached via a
linker. In some embodiments, the modified endogenous mammalian
protein comprises an antigen-binding domain, e.g., an antibody or
an antigen-binding antibody fragment. Exemplary, modified mammalian
proteins provided herein may comprise, e.g., single chain antibody,
camelid antibody, a VHH domain, a single-domain antibody, a
nanobody, an scFv, an adnectin, an affibody, an anticalin, an
aptamer, or a click chemistry handle. In some embodiments the
sortase recognition motif is positioned N-terminal with respect to
the endogenous polypeptide. In some embodiments the moiety is
attached to the N-terminal amino acid of the sortase recognition
motif or to an amino acid positioned N-terminal to the N-terminal
amino acid of the sortase recognition motif.
[0015] In some aspects, the present disclosure provides a method of
conjugating an agent to an animal cell, the method comprising:
contacting an animal cell with a sortase substrate that comprises a
sortase recognition sequence and an agent in the presence of a
sortase under conditions suitable for the sortase to conjugate the
sortase substrate to an endogenous, non-engineered polypeptide
expressed by the animal cell. In some embodiments the sortase
substrate is conjugated to an extracellular portion of an
endogenous, non-engineered polypeptide expressed by the cell. In
some embodiments the animal cell is a mammalian cell, e.g., a human
cell. In some embodiments the cell is an immune system cell, e.g.,
a lymphocyte (e.g., a T cell or NK cell), dendritic cell. In some
embodiments the cell is a cytotoxic cell. In some embodiments the
cell is a non-immortalized cell. In some embodiments the cell is a
primary cell. In some embodiments the animal cell is not
genetically engineered to express a polypeptide comprising a
sortase recognition sequence, a sequence comprising one or more
glycines, or both. In some embodiments the animal cell is not
genetically engineered to express a polypeptide comprising a
sortase recognition sequence, a sequence comprising one or more
alanines, or both. In some embodiments the animal cell is not
genetically engineered to express a polypeptide comprising a
sequence that renders the polypeptide usable in a sortase-catalyzed
reaction. In some embodiments, the animal cell is not chemically
engineered to present a polypeptide comprising a sortase
recognition sequence, a sequence comprising one or more glycines,
or both, on its surface. In some embodiments, the animal cell is
not chemically engineered to present a polypeptide comprising a
sortase recognition sequence, a sequence comprising one or more
alanines, or both, on its surface. In some embodiments the animal
cell is not chemically engineered to present at its surface a
moiety that renders the polypeptide usable in a sortase-catalyzed
reaction. In some embodiments the animal cell is not chemically
engineered to present at its surface a polypeptide comprising a
sequence that renders the polypeptide usable in a sortase-catalyzed
reaction. In some embodiments the cell is not stably or transiently
transfected or infected with a nucleic acid construct or vector
encoding a protein comprising a sortase recognition sequence,
nucleophilic acceptor sequence, or both. In some embodiments the
cell is not genetically engineered. In some embodiments the cell
originates from a subject in need of evaluation or treatment for a
disease of interest or from a donor who is immunocompatible with
the subject. In some embodiments the cell originates from a subject
in need of evaluation or treatment for a disease characterized by
the presence of abnormal or excessive cells or pathogens in the
subject's body or from a donor who is immunocompatible with the
subject. In some embodiments the cell originates from a subject in
need treatment for a disease characterized by deterioration or
dysfunction of a tissue or organ, wherein regenerative medicine
therapy may be useful. In some embodiments the cell originates from
a subject in need of evaluation or treatment for cancer, an
autoimmune disease, or an infection or from a donor who is
immunocompatible with the subject. In some embodiments the sortase
is a Sortase A, e.g., Staphylococcus aureus Sortase A. In some
embodiments the sortase recognition sequence comprises LPXTG. In
some embodiments the agent comprises an amino acid, a peptide, a
protein, a polynucleotide, a carbohydrate, a tag, a metal atom, a
chelating agent, a contrast agent, a catalyst, a polymer, a
recognition element, a small molecule, a lipid, a label, an
epitope, an antigen, a therapeutic agent, a cross-linker, a toxin,
a radioisotope, an antibody, an antibody domain, a click chemistry
handle, a virus, a cell, or a particle. In some embodiments the
agent comprises a targeting moiety that binds to an epitope or
antigen of interest. In some embodiments the targeting moiety binds
to a tumor antigen or a viral, bacterial, fungal, or parasite
antigen, or a cellular marker. In some embodiments the agent
comprises one or more of the following: (a) a targeting moiety, (b)
a costimulatory domain, (c) a signaling domain, (d) a receptor
domain, (e) an activating domain, (f) an antigen-binding portion of
an antigen receptor; (g) an enzyme; (h) a cytolytic domain; (i) a
pro-apoptotic domain. In some embodiments the method comprises
obtaining the cell or an ancestor of the animal cell from a subject
in need of evaluation or treatment for a disease of interest or
from a donor who is immunocompatible with the subject. In some
embodiments the method comprises separating the animal cell that
has the sortase substrate conjugated thereto from the sortase,
unconjugated sortase substrate, or both. In some embodiments the
method comprises detecting the agent conjugated to the animal cell.
In some embodiments the method comprises administering the animal
cell having the agent conjugated thereto to a subject. In some
aspects, the disclosure provides an isolated animal cell or
population of isolated animal cells prepared according to any of
the methods. In some embodiments the cell or population of cells is
suitable for administration to a human subject.
[0016] In some aspects, the disclosure provides an isolated animal
cell comprising an endogenous, non-engineered polypeptide
comprising a sortase recognition sequence that has an agent
conjugated thereto. In some embodiments the sortase substrate is
conjugated to an extracellular portion of an endogenous,
non-engineered polypeptide expressed by the cell. In some
embodiments the cell is a mammalian cell, e.g., a human cell. In
some embodiments the cell is an immune system cell, e.g., a
lymphocyte (e.g., a T cell), NK cell, dendritic cell. In some
embodiments the cell is a non-immortalized cell. In some
embodiments the cell is a primary cell. In some embodiments the
animal cell is not genetically engineered to express a polypeptide
comprising a sortase recognition sequence, a sequence comprising
one or more glycines, or both. In some embodiments the animal cell
is not genetically engineered to express a polypeptide comprising a
sortase recognition sequence, a sequence comprising one or more
alanines, or both. In some embodiments the cell is not genetically
engineered. In some embodiments the cell is not chemically
engineered. In some embodiments the cell originates from a subject
in need of evaluation or treatment for a disease of interest or
from a donor who is immunocompatible with the subject. In some
embodiments the cell originates from a subject in need of
evaluation or treatment for a disease characterized by the presence
of abnormal or excessive cells or pathogens in the subject's body
or from a donor who is immunocompatible with the subject. In some
embodiments the cell originates from a subject in need of
evaluation or treatment for cancer, an autoimmune disease, or an
infection or from a donor who is immunocompatible with the subject.
In some embodiments the sortase recognition sequence comprises
LPXTG. In some embodiments the agent comprises an amino acid, a
peptide, a protein, a polynucleotide, a carbohydrate, a tag, a
metal atom, a chelating agent, a contrast agent, a catalyst, a
polymer, a recognition element, a small molecule, a lipid, a label,
an epitope, an antigen, a therapeutic agent, a cross-linker, a
toxin, a radioisotope, an antibody, an antibody domain, a click
chemistry handle, a virus, a cell, or a particle. In some
embodiments the agent comprises a targeting moiety that binds to an
epitope or antigen of interest. In some embodiments the agent
comprises a targeting moiety that binds to a tumor antigen or a
viral, bacterial, fungal, or parasite antigen. In some embodiments
the agent comprises one or more of the following: (a) a targeting
moiety, (b) a costimulatory domain, (c) a signaling domain, (d) a
receptor domain, (e) an activating domain, (f) an antigen-binding
portion of an antigen receptor; (g) an enzyme; (h) a cytolytic
domain; (i) a pro-apoptotic domain. In some embodiments the agent
is detectable by fluorescence activated cell sorting (FACS),
fluorescence microscopy, Western blot, ELISA, chromatography, or
mass spectrometry after being conjugated to the cell.
[0017] In some aspects, the disclosure provides a method of
administering an agent to a subject comprising: (a) providing the
isolated animal cell or population of animal cells described herein
and (b) administering the isolated animal cell or population of
animal cells to the subject.
[0018] In some aspects, the disclosure provides composition
comprising: (i) an animal cell comprising an endogenous,
non-engineered polypeptide comprising a sequence capable as serving
as a nucleophile in a sortase-mediated reaction; (ii) a sortase
substrate comprising a sortase recognition motif; and (iii) a
sortase. In some embodiments the animal cell is a mammalian cell,
e.g., a human cell. In some embodiments the sortase is a sortase A.
In some embodiments the animal cell is an immune system cell, e.g.,
a lymphocyte (e.g., a T cell or NK cell) or dendritic cell. In some
embodiments the cell is a cytotoxic cell. In some embodiments the
cell is a non-immortalized cell. In some embodiments the cell is a
primary cell. In some embodiments the cell is not genetically
engineered to express a polypeptide comprising a sortase
recognition sequence, a sequence comprising one or more glycines,
or both. In some embodiments the cell is not genetically engineered
to express a polypeptide comprising a sortase recognition sequence,
a sequence comprising one or more alanines, or both. In some
embodiments the cell is not genetically engineered. In some
embodiments the cell is not chemically engineered. In some
embodiments the cell originates from a subject in need of
evaluation or treatment for a disease of interest or from a donor
who is immunocompatible with the subject. In some embodiments the
cell originates from a subject in need of evaluation or treatment
for a disease characterized by the presence of abnormal or
excessive cells or pathogens in the subject's body or from a
immunocompatible donor. In some embodiments the cell originates
from a subject in need of evaluation or treatment for cancer, an
autoimmune disease, or an infection or from a donor who is
immunocompatible with the subject. In some embodiments the sortase
is a Sortase A, e.g., Staphylococcus aureus Sortase A. In some
embodiments the sortase recognition sequence comprises LPXTG. In
some embodiments the agent comprises an amino acid, a peptide, a
protein, a polynucleotide, a carbohydrate, a tag, a metal atom, a
chelating agent, a contrast agent, a catalyst, a polymer, a
recognition element, a small molecule, a lipid, a label, an
epitope, an antigen, a therapeutic agent, a cross-linker, a toxin,
a radioisotope, an antibody, an antibody domain, a click chemistry
handle, a virus, a cell, or a particle. In some embodiments the
agent comprises a targeting moiety that binds to an epitope or
antigen of interest. In some embodiments the agent comprises a
targeting moiety that binds to a tumor antigen or a viral,
bacterial, fungal, or parasite antigen. In some embodiments the
agent comprises one or more of the following: (a) a targeting
moiety, (b) a costimulatory domain, (c) a signaling domain, (d) a
receptor domain, (e) an activating domain, (f) an antigen-binding
portion of an antigen receptor; (g) an enzyme; (h) a cytolytic
domain; (i) a pro-apoptotic domain. In some embodiments the cell
originates from a subject in need of evaluation or treatment for a
disease of interest or from a donor who is immunocompatible with
the subject.
[0019] In some aspects, the disclosure provides a method of
modulating an immune response of a subject to an entity of
interest, the method comprising administering to the subject an
animal cell that comprises an endogenous, non-engineered
polypeptide comprising a sortase recognition sequence that has an
agent conjugated thereto, wherein the agent comprises an antigen or
epitope of the entity of interest or a targeting moiety that binds
to an antigen or epitope of the entity of interest. In some
embodiments the cell is a mammalian cell, e.g., a human cell. In
some embodiments the cell is an immune system cell, e.g., a
lymphocyte (e.g., a T cell or NK cell) or dendritic cell. In some
embodiments the entity of interest is a cancer cell, an infected
cell, or a pathogen. In some embodiments the antigen is a tumor
antigen or a viral, bacterial, fungal, or parasite antigen. In some
embodiments modulating an immune response comprises stimulating an
immune response directed towards the entity of interest. In some
embodiments the entity of interest is a self cell or structure. In
some embodiments the entity of interest is an environmental
allergen. In some embodiments modulating an immune response
comprises inhibiting an immune response directed towards the entity
of interest. In some embodiments modulating an immune response
comprises increasing or inducing tolerance towards the entity of
interest. In some embodiments the agent comprises one or more of
the following: (a) a targeting moiety, (b) a costimulatory domain,
(c) a signaling domain, (d) a receptor domain, (e) an activating
domain, (f) an antigen-binding portion of an antigen receptor; (h)
a cytolytic domain; (i) a pro-apoptotic domain.
[0020] In some aspects, the disclosure provides a method of
neutralizing a substance in the body of a subject, the method
comprising administering to the subject an animal cell that
comprises an endogenous, non-engineered polypeptide comprising a
sortase recognition sequence that has an agent conjugated thereto,
wherein the agent binds to the substance. In some embodiments the
substance comprises a toxin. In some embodiments the substance
comprises an inflammatory cytokine. In some embodiments the agent
comprises an antibody or antigen-binding fragment thereof or a
portion of a receptor that binds to the substance.
[0021] In some aspects, the disclosure provides a method of
treating a subject in need of treatment for deficiency of a
protein, the method comprising administering to the subject an
animal cell that comprises an endogenous, non-engineered
polypeptide comprising a sortase recognition sequence that has an
agent conjugated thereto, wherein the agent comprises the protein.
In some embodiments the protein is an enzyme. In some embodiments
the protein is normally found in the blood.
[0022] In some aspects, the disclosure provides a method of
treating a subject in need of treatment for a disease, the method
comprising administering to the subject an animal cell that
comprises an endogenous, non-engineered polypeptide comprising a
sortase recognition sequence that has an agent conjugated thereto,
wherein the agent comprises a therapeutic agent effective for
treating the disease. In some embodiments the therapeutic agent
comprises a chemotherapy drug, anti-infective agent (e.g.,
antibacterial, antiviral, antifungal, or antiparasite agent),
enzyme, or monoclonal antibody. In some embodiments the cell is a
human cell. In some embodiments the cell originates from the
subject or from an immunocompatible donor.
[0023] In some embodiments of any method comprising administering a
cell to a subject, the subject is a human subject.
[0024] In some embodiments of any method comprising administering a
cell to a subject, the cell is a human cell (e.g., an autologous or
immunocompatible cell) and the subject is a human subject.
[0025] In some embodiments of any method comprising administering a
cell, the cell is administered into the circulatory system, e.g.,
intravenously.
[0026] The above summary is intended to give an overview over some
aspects of this invention, and is not to be construed to limit the
invention in any way. Additional aspects, advantages, and
embodiments of this invention are described herein, and further
embodiments will be apparent to those of skill in the art based on
the instant disclosure. The entire contents of all references cited
in this document are hereby incorporated by reference.
[0027] The practice of certain aspects of the present invention may
employ conventional techniques of molecular biology, cell culture,
recombinant nucleic acid (e.g., DNA) technology, immunology,
transgenic biology, microbiology, nucleic acid and polypeptide
synthesis, detection, manipulation, and quantification, and RNA
interference that are within the ordinary skill of the art. See,
e.g., Ausubel, F., et al., (eds.), Current Protocols in Molecular
Biology, Current Protocols in Immunology, Current Protocols in
Protein Science, and Current Protocols in Cell Biology, all John
Wiley & Sons, N.Y., edition as of December 2008 or more recent
editions; Sambrook, Russell, and Sambrook, Molecular Cloning: A
Laboratory Manual, .sup.3rd ed., Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, 2001; Harlow, E. and Lane, D.,
Antibodies--A Laboratory Manual, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, 1988. Information regarding various
diseases and diagnosis and certain treatments of such diseases is
found in Longo, D., et al. (eds.), Harrison's Principles of
Internal Medicine, 18th Edition; McGraw-Hill Professional, 2011.
Information regarding various therapeutic agents and human diseases
is found in Brunton, L., et al. (eds.) Goodman and Gilman's The
Pharmacological Basis of Therapeutics, 12.sup.th Ed., McGraw Hill,
2010 and/or Katzung, B. (ed.) Basic and Clinical Pharmacology,
McGraw-Hill/Appleton & Lange; 11th edition (July 2009).
Information regarding the immune system, immune system cells, and
proteins and other molecules produced by immune system cells and/or
that play a role in the immune system or immune response may be
found in standard immunology textbooks such as Paul, W E (ed.),
Fundamental Immunology, Lippincott Williams & Wilkins; 6th ed.,
2008; Murphy, K, Janeway's Immunobiology, 8th ed., Garland Science,
Taylor & Francis Group, London and New York (2012). All
patents, patent applications, books, articles, documents,
databases, websites, publications, references, etc., mentioned in
this document are incorporated by reference in their entirety. In
case of a conflict between the specification and any of the
incorporated references, the specification (including any
amendments thereof), shall control. Applicants reserve the right to
amend the specification based, e.g., on any of the incorporated
material and/or to correct obvious errors. None of the content of
the incorporated material shall limit the invention.
BRIEF DESCRIPTION OF THE DRAWING
[0028] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawings will be provided by the Office upon
request and payment of the necessary fee.
[0029] FIGS. 1A-1D. FIG. 1A. Schematic representation of a
sortase-catalyzed transacylation reaction in which a sortase
substrate protein is conjugated to a nucleophile, with release of a
portion of the substrate protein comprising an epitope tag. FIG.
1B. Schematic representation of sortase-catalyzed conjugation of
G(n)-Probe to the C-terminus of a LPETG-tagged protein using
sortase A. FIG. 1C. Schematic representation of sortase-catalyzed
conjugation of Probe-LPETG to the N-terminus to a G(n)-tagged
protein using sortase A. FIG. 1D. Schematic representation of
sortase-catalyzed conjugation of LPETG-tagged probe or protein to
naturally exposed N-terminal glycine residues at the surface of
cells. (G)n in FIGS. 1B-1D represents a sequence of one or more
glycines.
[0030] FIG. 2. Immunoblot demonstrating sortase-catalyzed
conjugation of biotin to non-genetically engineered mammalian
cells. The blot shows biotinylated proteins in cell lysate
following incubation of mouse red cell-depleted splenocytes with a
biotin-LPETG probe in the presence (right) or absence (left) of
sortase.
[0031] FIG. 3. Flow cytometry analysis demonstrating
sortase-catalyzed conjugation of biotin to non-genetically
engineered mammalian cells. Mouse red cell-depleted splenocytes
were incubated with biotin-LPETG probe with or without sortase,
washed with PBS, and incubated with phycoerythrin (PE)-conjugated
strepavidin. Blue histograms (indicated with arrows) show PE signal
gated on living cells incubated with (right) or without (left)
sortase A. Black histograms (no arrow) show background staining on
control splenocytes.
[0032] FIG. 4. Flow cytometry analysis demonostrating
sortase-catalyzed conjugation of a GFP-specific VHH to
non-genetically engineered mammalian cells. Mouse red cell-depleted
splenocytes were incubated with a GFP-specific VHH that contains a
C-terminal LPETG with or without sortase. Cells were then washed
with PBS and incubated with GFP. Blue histograms (indicated with
arrows) show GFP signal gated on living cells incubated with
(right) or without (left) sortase A. Black histograms (no arrow)
show background staining on control splenocytes.
[0033] FIGS. 5A-5F. FIGS. 5A-5D. Immunoblots demonstrating
sortase-catalyzed conjugation of a biotin-containing sortase
substrate to non-genetically engineered S. cerevesiae cells (FIG.
5A), T. gondii cells (FIG. 5B), HEK-293T cells (FIG. 5C), and mouse
splenocytes (FIG. 5D), as evidenced by streptavidin-based detection
of protein. FIG. 5E. Histograms showing flow cytometric analysis of
non-genetically engineered splenocytes that were sortagged with a
biotin-containing sortase substrate (biotin-LPETG) and subsequently
exposed to streptavidin-phycoerythrin (streptavidin PE). The
rightmost peak in each histogram represents streptavidin PE-labeled
splenocytes. FIG. 5F. Erythrocyte-depleted splenocytes were
incubated with 20 .mu.M sortase A and 500 .mu.M biotin-LPETG for
the indicated times. Cells were washed and incubated with
streptavidin-PE and analyzed by flow cytometry. Scatter plots show
the mean fluorescence intensity of streptavidin-PE staining for
each time point, normalized to maximum staining (120 minutes).
[0034] FIG. 6. Flow cytometry analysis demonstrating cytotoxicity
of red cell depleted splenocytes from OTI Rag-/-mice (which express
a T cell receptor specific for the SIINFELK peptide) towards
splenocytes that display SIINFEKL peptide at their cell
surface.
[0035] FIGS. 7A-7D. Installation of VHHs on activated CD8+ T cells
and demonstration of cytotoxicity of sortase-labeled CD8+ cells
towards target cells expressing VHH target antigen. In vitro
activated CD8+ T cells from OTI rag-/- mice were incubated for 1
hour at room temperature with or without 500 .mu.M, 50 .mu.M, or 5
.mu.M of enhancer-LPETG or VHH7-LPETG and with or without 20 .mu.M
of sortase A, as indicated. FIG. 7A. Control or sortagged cells
were incubated with purified GFP protein. Binding of GFP through
conjugated enhancer-LPETG was analyzed by flow cytometry. FIG. 7B.
Control or sortagged cells were incubated with purified GFP
protein. Amount of bound GFP was estimated by analyzing cell
lysates by SDS-PAGE and Western blotting against GFP protein and
comparing signal to a GFP standard (right lanes). FIGS. 7C-7D.
Control (FIG. 7C) or sortagged cells (FIG. 7D) were incubated with
splenocytes from WT mice for 20 hours. Histograms show the
percentage of propidium iodide negative CD4 and CD19 cells as
compared to cells incubated with unmanipulated activated OTI CD8 T
cells. Enhancer VHH is a VHH that binds to GFP. VHH7 is a VHH that
binds to MHC Class II but does not bind to GFP. Following
incubation, cells were washed, contacted with GFP, and subjected to
flow cytometry to detect GFP bound to the cells.
[0036] FIGS. 8A-8C. FIG. 8A. Toxoplasma gondii tachyzoites were
incubated with 500 .mu.M TAMRA-LPETG and 20 .mu.M sortase A for 15
minutes. Parasites were then washed and incubated with human
foreskin fibroblasts. Images show the juxtaposition of bright and
fluorescent fields. Black arrow: intracellular parasite. White
arrow: invading parasite. Scale bar: 10 micrometer. Right panels:
zoomed images. FIG. 8B. Histogram showing the percentage of
sortagged Toxoplasma positive cells within CD19 negative (light
gray bars) or CD19 positive (dark gray bars) splenocyte populations
after incubation of Enhancer-sortagged or VHH-7 sortagged
Toxoplasma gondii with mouse splenocytes. FIG. 8C. Purified B cells
from WT or class II MHC k.o. mice were incubated together with
control T. gondii or T. gondii sortagged with enhancer or VHH7 at a
multiplicity of infection of 5. Fifteen hours after infection, cell
lysis was measured and normalized to uninfected (0%) and
detergent-lysed B cells (100%). Error bars: standard deviation
(n=3). **: p<0.01 at Student T-test.
[0037] FIG. 9. Kinetic analysis showing installation of two
substrates: LPETG-biotin and single domain anti-GFP VHH (Enhancer)
on cells as a function of time at 37 degrees C.
[0038] FIG. 10. Installation of LPETG-biotin on intact cells,
followed by staining with streptavidin-PE. Panels show reaction
with no enzyme (left panel), S. aureus Sortase A (middle panel) and
Ca.sup.2+-independent sortase heptamutant (right panel) at the
indicated temperatures (bottom right). Mutations in heptamutant are
shown in SEQ ID NOs: 4 and 7.
[0039] FIG. 11. Sequential installation of Enhancer first, then
installation of LPETG-biotin, demonstrating that there are
remaining sites on the cells that can serve as nucleophile to
accept LPETG on cells already modified with Enhancer.
Erythrocyte-depleted splenocytes were incubated with or without 500
.mu.M enhancer-LPETG and 20 .mu.M sortase A. After 60 minutes, 500
.mu.M biotin-LPETG was added to reactions where indicated for a
further 15 minutes. Dot plots show the binding of APC-conjugated
streptavidin and GFP by sortagged cells after washing.
DEFINITIONS
[0040] Definitions of specific functional groups and chemical terms
are described in more detail below. For purposes of this invention,
the chemical elements are identified in accordance with the
Periodic Table of the Elements, CAS version, Handbook of Chemistry
and Physics, 75th Ed., inside cover, and specific functional groups
are generally defined as described therein. Additionally, general
principles of organic chemistry, as well as specific functional
moieties and reactivity, are described in Organic Chemistry, Thomas
Sorrell, University Science Books, Sausalito, 1999; Smith and March
March's Advanced Organic Chemistry, 5th Edition, John Wiley &
Sons, Inc., New York, 2001; Larock, Comprehensive Organic
Transformations, VCH Publishers, Inc., New York, 1989; Carruthers,
Some Modern Methods of Organic Synthesis, 3rd Edition, Cambridge
University Press, Cambridge, 1987.
[0041] The term "aliphatic," as used herein, includes both
saturated and unsaturated, nonaromatic, straight chain (i.e.,
unbranched), branched, acyclic, and cyclic (i.e., carbocyclic)
hydrocarbons, which are optionally substituted with one or more
functional groups. As will be appreciated by one of ordinary skill
in the art, "aliphatic" is intended herein to include, but is not
limited to, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and
cycloalkynyl moieties. Thus, as used herein, the term "alkyl"
includes straight, branched and cyclic alkyl groups. An analogous
convention applies to other generic terms such as "alkenyl,"
"alkynyl," and the like. Furthermore, as used herein, the terms
"alkyl," "alkenyl," "alkynyl," and the like encompass both
substituted and unsubstituted groups. In certain embodiments, as
used herein, "aliphatic" is used to indicate those aliphatic groups
(cyclic, acyclic, substituted, unsubstituted, branched or
unbranched) having 1-20 carbon atoms (C.sub.1-20 aliphatic). In
certain embodiments, the aliphatic group has 1-10 carbon atoms
(C.sub.1-10 aliphatic). In certain embodiments, the aliphatic group
has 1-6 carbon atoms (C.sub.1-6 aliphatic). In certain embodiments,
the aliphatic group has 1-5 carbon atoms (C.sub.1-5 aliphatic). In
certain embodiments, the aliphatic group has 1-4 carbon atoms
(C.sub.1-4 aliphatic). In certain embodiments, the aliphatic group
has 1-3 carbon atoms (C.sub.1-3 aliphatic). In certain embodiments,
the aliphatic group has 1-2 carbon atoms (C.sub.1-2 aliphatic).
Aliphatic group substituents include, but are not limited to, any
of the substituents described herein, that result in the formation
of a stable moiety.
[0042] The term "alkyl," as used herein, refers to saturated,
straight- or branched-chain hydrocarbon radicals derived from a
hydrocarbon moiety containing between one and twenty carbon atoms
by removal of a single hydrogen atom. In some embodiments, the
alkyl group employed in the invention contains 1-20 carbon atoms
(C.sub.1-20 alkyl). In another embodiment, the alkyl group employed
contains 1-15 carbon atoms (C.sub.1-15alkyl). In another
embodiment, the alkyl group employed contains 1-10 carbon atoms
(C.sub.1-10alkyl). In another embodiment, the alkyl group employed
contains 1-8 carbon atoms (C.sub.1-8 alkyl). In another embodiment,
the alkyl group employed contains 1-6 carbon atoms (C.sub.1-6
alkyl). In another embodiment, the alkyl group employed contains
1-5 carbon atoms (C.sub.1-5alkyl). In another embodiment, the alkyl
group employed contains 1-4 carbon atoms (C.sub.1-4alkyl). In
another embodiment, the alkyl group employed contains 1-3 carbon
atoms (C.sub.1-3 alkyl). In another embodiment, the alkyl group
employed contains 1-2 carbon atoms (C.sub.1-2 alkyl). Examples of
alkyl radicals include, but are not limited to, methyl, ethyl,
n-propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, sec-pentyl,
iso-pentyl, tert-butyl, n-pentyl, neopentyl, n-hexyl, sec-hexyl,
n-heptyl, n-octyl, n-decyl, n-undecyl, dodecyl, and the like, which
may bear one or more substituents. Alkyl group substituents
include, but are not limited to, any of the substituents described
herein, that result in the formation of a stable moiety. The term
"alkylene," as used herein, refers to a biradical derived from an
alkyl group, as defined herein, by removal of two hydrogen atoms.
Alkylene groups may be cyclic or acyclic, branched or unbranched,
substituted or unsubstituted. Alkylene group substituents include,
but are not limited to, any of the substituents described herein,
that result in the formation of a stable moiety.
[0043] The term "alkenyl," as used herein, denotes a monovalent
group derived from a straight- or branched-chain hydrocarbon moiety
having at least one carbon-carbon double bond by the removal of a
single hydrogen atom. In certain embodiments, the alkenyl group
employed in the invention contains 2-20 carbon atoms (C.sub.2-20
alkenyl). In some embodiments, the alkenyl group employed in the
invention contains 2-15 carbon atoms (C.sub.2-15 alkenyl). In
another embodiment, the alkenyl group employed contains 2-10 carbon
atoms (C.sub.2-10 alkenyl). In still other embodiments, the alkenyl
group contains 2-8 carbon atoms (C.sub.2-8 alkenyl). In yet other
embodiments, the alkenyl group contains 2-6 carbons (C.sub.2-6
alkenyl). In yet other embodiments, the alkenyl group contains 2-5
carbons (C.sub.2-5 alkenyl). In yet other embodiments, the alkenyl
group contains 2-4 carbons (C.sub.2-4 alkenyl). In yet other
embodiments, the alkenyl group contains 2-3 carbons (C.sub.2-3
alkenyl). In yet other embodiments, the alkenyl group contains 2
carbons (C.sub.2alkenyl). Alkenyl groups include, for example,
ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, and the like,
which may bear one or more substituents. Alkenyl group substituents
include, but are not limited to, any of the substituents described
herein, that result in the formation of a stable moiety. The term
"alkenylene," as used herein, refers to a biradical derived from an
alkenyl group, as defined herein, by removal of two hydrogen atoms.
Alkenylene groups may be cyclic or acyclic, branched or unbranched,
substituted or unsubstituted. Alkenylene group substituents
include, but are not limited to, any of the substituents described
herein, that result in the formation of a stable moiety.
[0044] The term "alkynyl," as used herein, refers to a monovalent
group derived from a straight- or branched-chain hydrocarbon having
at least one carbon-carbon triple bond by the removal of a single
hydrogen atom. In certain embodiments, the alkynyl group employed
in the invention contains 2-20 carbon atoms (C.sub.2-20 alkynyl).
In some embodiments, the alkynyl group employed in the invention
contains 2-15 carbon atoms (C.sub.2-15 alkynyl). In another
embodiment, the alkynyl group employed contains 2-10 carbon atoms
(C.sub.2-10 alkynyl). In still other embodiments, the alkynyl group
contains 2-8 carbon atoms (C.sub.2-8 alkynyl). In still other
embodiments, the alkynyl group contains 2-6 carbon atoms (C.sub.2-6
alkynyl). In still other embodiments, the alkynyl group contains
2-5 carbon atoms (C.sub.2-5 alkynyl). In still other embodiments,
the alkynyl group contains 2-4 carbon atoms (C.sub.2-4 alkynyl). In
still other embodiments, the alkynyl group contains 2-3 carbon
atoms (C.sub.2-3 alkynyl). In still other embodiments, the alkynyl
group contains 2 carbon atoms (C.sub.2alkynyl). Representative
alkynyl groups include, but are not limited to, ethynyl, 2-propynyl
(propargyl), 1-propynyl, and the like, which may bear one or more
substituents. Alkynyl group substituents include, but are not
limited to, any of the substituents described herein, that result
in the formation of a stable moiety. The term "alkynylene," as used
herein, refers to a biradical derived from an alkynylene group, as
defined herein, by removal of two hydrogen atoms. Alkynylene groups
may be cyclic or acyclic, branched or unbranched, substituted or
unsubstituted. Alkynylene group substituents include, but are not
limited to, any of the substituents described herein, that result
in the formation of a stable moiety.
[0045] The term "carbocyclic" or "carbocyclyl" as used herein,
refers to an as used herein, refers to a cyclic aliphatic group
containing 3-10 carbon ring atoms (C.sub.3-10carbocyclic).
Carbocyclic group substituents include, but are not limited to, any
of the substituents described herein, that result in the formation
of a stable moiety.
[0046] The term "heteroaliphatic," as used herein, refers to an
aliphatic moiety, as defined herein, which includes both saturated
and unsaturated, nonaromatic, straight chain (i.e., unbranched),
branched, acyclic, cyclic (i.e., heterocyclic), or polycyclic
hydrocarbons, which are optionally substituted with one or more
functional groups, and that further contains one or more
heteroatoms (e.g., oxygen, sulfur, nitrogen, phosphorus, or silicon
atoms) between carbon atoms. In certain embodiments,
heteroaliphatic moieties are substituted by independent replacement
of one or more of the hydrogen atoms thereon with one or more
substituents. As will be appreciated by one of ordinary skill in
the art, "heteroaliphatic" is intended herein to include, but is
not limited to, heteroalkyl, heteroalkenyl, heteroalkynyl,
heterocycloalkyl, heterocycloalkenyl, and heterocycloalkynyl
moieties. Thus, the term "heteroaliphatic" includes the terms
"heteroalkyl," "heteroalkenyl," "heteroalkynyl," and the like.
Furthermore, as used herein, the terms "heteroalkyl,"
"heteroalkenyl," "heteroalkynyl," and the like encompass both
substituted and unsubstituted groups. In certain embodiments, as
used herein, "heteroaliphatic" is used to indicate those
heteroaliphatic groups (cyclic, acyclic, substituted,
unsubstituted, branched or unbranched) having 1-20 carbon atoms and
1-6 heteroatoms (C.sub.1-20 heteroaliphatic). In certain
embodiments, the heteroaliphatic group contains 1-10 carbon atoms
and 1-4 heteroatoms (C.sub.1-10heteroaliphatic). In certain
embodiments, the heteroaliphatic group contains 1-6 carbon atoms
and 1-3 heteroatoms (C.sub.1-6heteroaliphatic). In certain
embodiments, the heteroaliphatic group contains 1-5 carbon atoms
and 1-3 heteroatoms (C.sub.1-5 heteroaliphatic). In certain
embodiments, the heteroaliphatic group contains 1-4 carbon atoms
and 1-2 heteroatoms (C.sub.1-4 heteroaliphatic). In certain
embodiments, the heteroaliphatic group contains 1-3 carbon atoms
and 1 heteroatom (C.sub.1-3heteroaliphatic). In certain
embodiments, the heteroaliphatic group contains 1-2 carbon atoms
and 1 heteroatom (C.sub.1-2 heteroaliphatic). Heteroaliphatic group
substituents include, but are not limited to, any of the
substituents described herein, that result in the formation of a
stable moiety.
[0047] The term "heteroalkyl," as used herein, refers to an alkyl
moiety, as defined herein, which contain one or more heteroatoms
(e.g., oxygen, sulfur, nitrogen, phosphorus, or silicon atoms) in
between carbon atoms. In certain embodiments, the heteroalkyl group
contains 1-20 carbon atoms and 1-6 heteroatoms (C.sub.1-20
heteroalkyl). In certain embodiments, the heteroalkyl group
contains 1-10 carbon atoms and 1-4 heteroatoms (C.sub.1-10
heteroalkyl). In certain embodiments, the heteroalkyl group
contains 1-6 carbon atoms and 1-3 heteroatoms (C.sub.1-6
heteroalkyl). In certain embodiments, the heteroalkyl group
contains 1-5 carbon atoms and 1-3 heteroatoms (C.sub.1-5
heteroalkyl). In certain embodiments, the heteroalkyl group
contains 1-4 carbon atoms and 1-2 heteroatoms (C.sub.1-4
heteroalkyl). In certain embodiments, the heteroalkyl group
contains 1-3 carbon atoms and 1 heteroatom (C.sub.1-3 heteroalkyl).
In certain embodiments, the heteroalkyl group contains 1-2 carbon
atoms and 1 heteroatom (C.sub.1-2 heteroalkyl). The term
"heteroalkylene," as used herein, refers to a biradical derived
from an heteroalkyl group, as defined herein, by removal of two
hydrogen atoms. Heteroalkylene groups may be cyclic or acyclic,
branched or unbranched, substituted or unsubstituted.
Heteroalkylene group substituents include, but are not limited to,
any of the substituents described herein, that result in the
formation of a stable moiety.
[0048] The term "heteroalkenyl," as used herein, refers to an
alkenyl moiety, as defined herein, which further contains one or
more heteroatoms (e.g., oxygen, sulfur, nitrogen, phosphorus, or
silicon atoms) in between carbon atoms. In certain embodiments, the
heteroalkenyl group contains 2-20 carbon atoms and 1-6 heteroatoms
(C.sub.2-20 heteroalkenyl). In certain embodiments, the
heteroalkenyl group contains 2-10 carbon atoms and 1-4 heteroatoms
(C.sub.2-10 heteroalkenyl). In certain embodiments, the
heteroalkenyl group contains 2-6 carbon atoms and 1-3 heteroatoms
(C.sub.2-6 heteroalkenyl). In certain embodiments, the
heteroalkenyl group contains 2-5 carbon atoms and 1-3 heteroatoms
(C.sub.2-5 heteroalkenyl). In certain embodiments, the
heteroalkenyl group contains 2-4 carbon atoms and 1-2 heteroatoms
(C.sub.2-4 heteroalkenyl). In certain embodiments, the
heteroalkenyl group contains 2-3 carbon atoms and 1 heteroatom
(C.sub.2-3 heteroalkenyl). The term "heteroalkenylene," as used
herein, refers to a biradical derived from an heteroalkenyl group,
as defined herein, by removal of two hydrogen atoms.
Heteroalkenylene groups may be cyclic or acyclic, branched or
unbranched, substituted or unsubstituted.
[0049] The term "heteroalkynyl," as used herein, refers to an
alkynyl moiety, as defined herein, which further contains one or
more heteroatoms (e.g., oxygen, sulfur, nitrogen, phosphorus, or
silicon atoms) in between carbon atoms. In certain embodiments, the
heteroalkynyl group contains 2-20 carbon atoms and 1-6 heteroatoms
(C.sub.2-20 heteroalkynyl). In certain embodiments, the
heteroalkynyl group contains 2-10 carbon atoms and 1-4 heteroatoms
(C.sub.2-10 heteroalkynyl). In certain embodiments, the
heteroalkynyl group contains 2-6 carbon atoms and 1-3 heteroatoms
(C.sub.2-6 heteroalkynyl). In certain embodiments, the
heteroalkynyl group contains 2-5 carbon atoms and 1-3 heteroatoms
(C.sub.2-5 heteroalkynyl). In certain embodiments, the
heteroalkynyl group contains 2-4 carbon atoms and 1-2 heteroatoms
(C.sub.2-4 heteroalkynyl). In certain embodiments, the
heteroalkynyl group contains 2-3 carbon atoms and 1 heteroatom
(C.sub.2-3 heteroalkynyl). The term "heteroalkynylene," as used
herein, refers to a biradical derived from an heteroalkynyl group,
as defined herein, by removal of two hydrogen atoms.
Heteroalkynylene groups may be cyclic or acyclic, branched or
unbranched, substituted or unsubstituted.
[0050] The term "heterocyclic," "heterocycles," or "heterocyclyl,"
as used herein, refers to a cyclic heteroaliphatic group. A
heterocyclic group refers to a non-aromatic, partially unsaturated
or fully saturated, 3-to 10-membered ring system, which includes
single rings of 3 to 8 atoms in size, and bi- and tri-cyclic ring
systems which may include aromatic five- or six-membered aryl or
heteroaryl groups fused to a non-aromatic ring. These heterocyclic
rings include those having from one to three heteroatoms
independently selected from oxygen, sulfur, and nitrogen, in which
the nitrogen and sulfur heteroatoms may optionally be oxidized and
the nitrogen heteroatom may optionally be quaternized. In certain
embodiments, the term heterocyclic refers to a non-aromatic 5-, 6-,
or 7-membered ring or polycyclic group wherein at least one ring
atom is a heteroatom selected from O, S, and N (wherein the
nitrogen and sulfur heteroatoms may be optionally oxidized), and
the remaining ring atoms are carbon, the radical being joined to
the rest of the molecule via any of the ring atoms. Heterocycyl
groups include, but are not limited to, a bi- or tri-cyclic group,
comprising fused five, six, or seven-membered rings having between
one and three heteroatoms independently selected from the oxygen,
sulfur, and nitrogen, wherein (i) each 5-membered ring has 0 to 2
double bonds, each 6-membered ring has 0 to 2 double bonds, and
each 7-membered ring has 0 to 3 double bonds, (ii) the nitrogen and
sulfur heteroatoms may be optionally oxidized, (iii) the nitrogen
heteroatom may optionally be quaternized, and (iv) any of the above
heterocyclic rings may be fused to an aryl or heteroaryl ring.
Exemplary heterocycles include azacyclopropanyl, azacyclobutanyl,
1,3-diazatidinyl, piperidinyl, piperazinyl, azocanyl, thiaranyl,
thietanyl, tetrahydrothiophenyl, dithiolanyl, thiacyclohexanyl,
oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropuranyl, dioxanyl,
oxathiolanyl, morpholinyl, thioxanyl, tetrahydronaphthyl, and the
like, which may bear one or more substituents. Substituents
include, but are not limited to, any of the substituents described
herein, that result in the formation of a stable moiety.
[0051] The term "aryl," as used herein, refers to an aromatic mono-
or polycyclic ring system having 3-20 ring atoms, of which all the
ring atoms are carbon, and which may be substituted or
unsubstituted. In certain embodiments of the present invention,
"aryl" refers to a mono, bi, or tricyclic C.sub.4-C.sub.20 aromatic
ring system having one, two, or three aromatic rings which include,
but are not limited to, phenyl, biphenyl, naphthyl, and the like,
which may bear one or more substituents. Aryl substituents include,
but are not limited to, any of the substituents described herein,
that result in the formation of a stable moiety. The term
"arylene," as used herein refers to an aryl biradical derived from
an aryl group, as defined herein, by removal of two hydrogen atoms.
Arylene groups may be substituted or unsubstituted. Arylene group
substituents include, but are not limited to, any of the
substituents described herein, that result in the formation of a
stable moiety. Additionally, arylene groups may be incorporated as
a linker group into an alkylene, alkenylene, alkynylene,
heteroalkylene, heteroalkenylene, or heteroalkynylene group, as
defined herein.
[0052] The term "heteroaryl," as used herein, refers to an aromatic
mono- or polycyclic ring system having 3-20 ring atoms, of which
one ring atom is selected from S, O, and N; zero, one, or two ring
atoms are additional heteroatoms independently selected from S, O,
and N; and the remaining ring atoms are carbon, the radical being
joined to the rest of the molecule via any of the ring atoms.
Exemplary heteroaryls include, but are not limited to pyrrolyl,
pyrazolyl, imidazolyl, pyridinyl, pyrimidinyl, pyrazinyl,
pyridazinyl, triazinyl, tetrazinyl, pyyrolizinyl, indolyl,
quinolinyl, isoquinolinyl, benzoimidazolyl, indazolyl, quinolinyl,
isoquinolinyl, quinolizinyl, cinnolinyl, quinazolynyl,
phthalazinyl, naphthridinyl, quinoxalinyl, thiophenyl,
thianaphthenyl, furanyl, benzofuranyl, benzothiazolyl, thiazolynyl,
isothiazolyl, thiadiazolynyl, oxazolyl, isoxazolyl, oxadiaziolyl,
oxadiaziolyl, and the like, which may bear one or more
substituents. Heteroaryl substituents include, but are not limited
to, any of the substituents described herein, that result in the
formation of a stable moiety. The term "heteroarylene," as used
herein, refers to a biradical derived from an heteroaryl group, as
defined herein, by removal of two hydrogen atoms. Heteroarylene
groups may be substituted or unsubstituted. Additionally,
heteroarylene groups may be incorporated as a linker group into an
alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene,
or heteroalkynylene group, as defined herein. Heteroarylene group
substituents include, but are not limited to, any of the
substituents described herein, that result in the formation of a
stable moiety.
[0053] The term "acyl," as used herein, is a subset of a
substituted alkyl group, and refers to a group having the general
formula --C(.dbd.O)R.sup.A, --C(.dbd.O)OR.sup.A,
--C(.dbd.O)--O--C(.dbd.O)R.sup.A, --C(.dbd.O)SR.sup.A,
--C(.dbd.O)N(R.sup.A).sub.2, --C(.dbd.S)R.sup.A,
--C(.dbd.S)N(R.sup.A).sub.2, and --C(.dbd.S)S(R.sup.A),
--C(.dbd.NR.sup.A)R.sup.A, --C(.dbd.NR.sup.A)OR.sup.A,
--C(.dbd.NR.sup.A)SR.sup.A, and --C(.dbd.NR.sup.A)N(R.sup.A).sub.2,
wherein R.sup.A is hydrogen; halogen; substituted or unsubstituted
hydroxyl; substituted or unsubstituted thiol; substituted or
unsubstituted amino; acyl; optionally substituted aliphatic;
optionally substituted heteroaliphatic; optionally substituted
alkyl; optionally substituted alkenyl; optionally substituted
alkynyl; optionally substituted aryl, optionally substituted
heteroaryl, aliphaticoxy, heteroaliphaticoxy, alkyloxy,
heteroalkyloxy, aryloxy, heteroaryloxy, aliphaticthioxy,
heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy,
heteroarylthioxy, mono- or di-aliphaticamino, mono- or
di-heteroaliphaticamino, mono- or di-alkylamino, mono- or
di-heteroalkylamino, mono- or di-arylamino, or mono- or
di-heteroarylamino; or two R.sup.A groups taken together form a 5-
to 6-membered heterocyclic ring. Exemplary acyl groups include
aldehydes (--CHO), carboxylic acids (--CO.sub.2H), ketones, acyl
halides, esters, amides, imines, carbonates, carbamates, and ureas.
Acyl substituents include, but are not limited to, any of the
substituents described herein, that result in the formation of a
stable moiety.
[0054] The term "acylene," as used herein, is a subset of a
substituted alkylene, substituted alkenylene, substituted
alkynylene, substituted heteroalkylene, substituted
heteroalkenylene, or substituted heteroalkynylene group, and refers
to an acyl group having the general formulae:
--R.sup.0--(C.dbd.X.sup.1)--R.sup.0--,
--R.sup.0--X.sup.2(C.dbd.X.sup.1)--R.sup.0--, or
--R.sup.0--X.sup.2(C.dbd.X.sup.1)X.sup.3--R.sup.0--, where X.sup.1,
X.sup.2, and X.sup.3 is, independently, oxygen, sulfur, or
NR.sup.r, wherein R.sup.r is hydrogen or optionally substituted
aliphatic, and R.sup.0 is an optionally substituted alkylene,
alkenylene, alkynylene, heteroalkylene, heteroalkenylene, or
heteroalkynylene group, as defined herein. Exemplary acylene groups
wherein R.sup.0 is alkylene includes
--(CH.sub.2).sub.T--O(C.dbd.O)--(CH.sub.2).sub.T--;
--(CH.sub.2).sub.T--NR.sup.r(C.dbd.O)--(CH.sub.2).sub.T--;
--(CH.sub.2).sub.T--O(C.dbd.NR.sup.r)--(CH.sub.2).sub.T--;
--(CH.sub.2).sub.T--NR.sup.r(C.dbd.NR.sup.r)--(CH.sub.2).sub.T--;
--(CH.sub.2).sub.T--(C.dbd.O)--(CH.sub.2).sub.T--;
--(CH.sub.2).sub.T--(C.dbd.NR.sup.r)--(CH.sub.2).sub.T--;
--(CH.sub.2).sub.T--S(C.dbd.S)--(CH.sub.2).sub.T--;
--(CH.sub.2).sub.T--NR.sup.r(C.dbd.S)--(CH.sub.2).sub.T--;
--(CH.sub.2).sub.T--S(C.dbd.NR.sup.r)--(CH.sub.2).sub.T--;
--(CH.sub.2).sub.T--O(C.dbd.S)--(CH.sub.2).sub.T--;
--(CH.sub.2).sub.T--(C.dbd.S)--(CH.sub.2).sub.T--; or
--(CH.sub.2).sub.T--S(C.dbd.O)--(CH.sub.2).sub.T--, and the like,
which may bear one or more substituents; and wherein each instance
of T is, independently, an integer between 0 to 20. Acylene
substituents include, but are not limited to, any of the
substituents described herein, that result in the formation of a
stable moiety.
[0055] The term "amino," as used herein, refers to a group of the
formula (--NH.sub.2). A "substituted amino" refers either to a
mono-substituted amine (--NHR.sup.h) of a disubstituted amine
(--NR.sup.h.sub.2), wherein the R.sup.h substituent is any
substituent as described herein that results in the formation of a
stable moiety (e.g., an amino protecting group; aliphatic, alkyl,
alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl,
acyl, amino, nitro, hydroxyl, thiol, halo, aliphaticamino,
heteroaliphaticamino, alkylamino, heteroalkylamino, arylamino,
heteroarylamino, alkylaryl, arylalkyl, aliphaticoxy,
heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy,
heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy,
heteroalkylthioxy, arylthioxy, heteroarylthioxy, acyloxy, and the
like, each of which may or may not be further substituted). In
certain embodiments, the R.sup.h substituents of the di-substituted
amino group (--NR.sup.h.sub.2) form a 5- to 6-membered heterocyclic
ring.
[0056] The term "hydroxy" or "hydroxyl," as used herein, refers to
a group of the formula (--OH). A "substituted hydroxyl" refers to a
group of the formula (--OR.sup.i), wherein R.sup.i can be any
substituent which results in a stable moiety (e.g., a hydroxyl
protecting group; aliphatic, alkyl, alkenyl, alkynyl,
heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, nitro,
alkylaryl, arylalkyl, and the like, each of which may or may not be
further substituted).
[0057] The term "thio" or "thiol," as used herein, refers to a
group of the formula (--SH). A "substituted thiol" refers to a
group of the formula (--SR.sup.r), wherein R.sup.r can be any
substituent that results in the formation of a stable moiety (e.g.,
a thiol protecting group; aliphatic, alkyl, alkenyl, alkynyl,
heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, sulfinyl,
sulfonyl, cyano, nitro, alkylaryl, arylalkyl, and the like, each of
which may or may not be further substituted).
[0058] The term "imino," as used herein, refers to a group of the
formula (.dbd.NR.sup.r), wherein W corresponds to hydrogen or any
substituent as described herein, that results in the formation of a
stable moiety (for example, an amino protecting group; aliphatic,
alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl,
heteroaryl, acyl, amino, hydroxyl, alkylaryl, arylalkyl, and the
like, each of which may or may not be further substituted).
[0059] The term "azide" or "azido," as used herein, refers to a
group of the formula (--N.sub.3).
[0060] The terms "halo" and "halogen," as used herein, refer to an
atom selected from fluorine (fluoro, --F), chlorine (chloro, --Cl),
bromine (bromo, --Br), and iodine (iodo, --I).
[0061] A "leaving group" is an art-understood term referring to a
molecular fragment that departs with a pair of electrons in
heterolytic bond cleavage, wherein the molecular fragment is an
anion or neutral molecule. See, for example, Smith, March's
Advanced Organic Chemistry 6th ed. (501-502). Exemplary leaving
groups include, but are not limited to, halo (e.g., chloro, bromo,
iodo) and activated substituted hydroxyl groups, e.g., of the
formula --OC(.dbd.O)SR.sup.aa, --OC(.dbd.O)R.sup.aa,
--OCO.sub.2R.sup.aa, --OC(.dbd.O)N(R.sup.bb).sub.2,
--OC(.dbd.NR.sup.bb)R.sup.aa, --OC(.dbd.NR.sup.bb)OR.sup.aa,
--OC(.dbd.NR.sup.bb)N(R.sup.bb).sub.2, --OS(.dbd.O)R.sup.aa,
--OSO.sub.2R.sup.aa, --OP(R.sup.cc).sub.2, --OP(R.sup.cc).sub.3,
--OP(.dbd.O).sub.2R.sup.aa, --OP(.dbd.O)(R.sup.aa).sub.2,
--OP(.dbd.O)(OR.sup.cc).sub.2, --OP(.dbd.O).sub.2N(R.sup.bb).sub.2,
or --OP(.dbd.O)(NR.sup.bb).sub.2 wherein R.sup.aa is optionally
substituted aliphatic, optionally substituted heteroaliphatic,
optionally substituted aryl, or optionally substituted heteroaryl;
R.sup.bb is hydrogen, an amino protecting group, optionally
substituted aliphatic, optionally substituted heteroaliphatic,
optionally substituted aryl, or optionally substituted heteroaryl;
and R.sup.cc is hydrogen, optionally substituted aliphatic,
optionally substituted heteroaliphatic, optionally substituted
aryl, or optionally substituted heteroaryl.
[0062] The term "agent," as used herein, refers to any molecule,
entity, or moiety that can be conjugated to a sortase recognition
motif. For example, an agent may be a protein, an amino acid, a
peptide, a polynucleotide, a carbohydrate, a detectable label, a
binding agent, a tag, a metal atom, a contrast agent, a catalyst, a
non-polypeptide polymer, a synthetic polymer, a recognition
element, a lipid, a linker, or chemical compound, such as a small
molecule. In some embodiments, the agent is a binding agent, for
example, a ligand or a ligand-binding molecule, streptavidin,
biotin, an antibody or an antibody fragment. In some embodiments,
the agent cannot be genetically encoded. In some such embodiments,
the agent is a lipid, a carbohydrate, or a small molecule.
Additional agents suitable for use in embodiments of the present
invention will be apparent to the skilled artisan. The invention is
not limited in this respect.
[0063] The term "amino acid," as used herein, includes any
naturally occurring and nonnaturally occurring amino acid. Amino
acids include without limitation, natural alpha-amino acids such as
the 20 common naturally occurring alpha-amino acids found in
polypeptides and proteins (A, R, N, C, D, Q, E, G, H, I, L, K, M,
F, P, S, T, W, Y, V, also referred to as standard amino acids),
non-standard alpha-amino acids, and beta-amino acids. There are
many known non-standard, e.g., non-natural, amino acids any of
which may be included in the polypeptides or proteins described
herein. See, for example, S. Hunt, The Non-Protein Amino Acids in
Chemistry and Biochemistry of the Amino Acids, edited by G. C.
Barrett, Chapman and Hall, 1985 and/or Hughes, B. (ed.), Amino
Acids, Peptides and Proteins in Organic Chemistry, Volumes 1-4,
Wiley-VCH (2009-2011); Blaskovich, M., Handbook on Syntheses of
Amino Acids General Routes to Amino Acids, Oxford University Press,
2010. As used herein in the context of amino acid sequences, the
term X or Xaa represents any amino acid residue, e.g., any
naturally occurring and/or any non-naturally occurring amino acid
residue.
[0064] The term "binding agent" or "binding moiety" as used herein
refers to any molecule or entity that binds another molecule or
entity with high affinity. In some embodiments, a binding agent
binds its binding partner with high specificity. Examples of
binding agents include, without limitation, antibodies, antibody
fragments, receptors, ligands, aptamers, and adnectins.
[0065] The term "click chemistry" refers to a chemical philosophy
introduced by K. Barry Sharpless of The Scripps Research Institute,
describing chemistry tailored to generate covalent bonds quickly
and reliably by joining small units comprising reactive groups
together. Click chemistry does not refer to a specific reaction,
but to a concept including, but not limited to, reactions that
mimic reactions found in nature. In some embodiments, click
chemistry reactions are modular, wide in scope, give high chemical
yields, generate inoffensive byproducts, are stereospecific,
exhibit a large thermodynamic driving force>84 kJ/mol to favor a
reaction with a single reaction product, and/or can be carried out
under physiological conditions. In some embodiments, a click
chemistry reaction exhibits high atom economy, can be carried out
under simple reaction conditions, use readily available starting
materials and reagents, uses no toxic solvents or use a solvent
that is benign or easily removed (preferably water), and/or
provides simple product isolation by non-chromatographic methods
(crystallisation or distillation).
[0066] The term "click chemistry handle," as used herein, refers to
a reactant, or a reactive group, that can partake in a click
chemistry reaction. For example, a strained alkyne, e.g., a
cyclooctyne, is a click chemistry handle, since it can partake in a
strain-promoted cycloaddition (see, e.g., Table 1). In general,
click chemistry reactions require at least two molecules comprising
click chemistry handles that can react with each other. Such click
chemistry handle pairs that are reactive with each other are
sometimes referred to herein as partner click chemistry handles.
For example, an azide is a partner click chemistry handle to a
cyclooctyne or any other alkyne. Exemplary click chemistry handles
suitable for use according to some aspects of this invention are
described herein, for example, in Tables 1 and 2. Other suitable
click chemistry handles are known to those of skill in the art.
[0067] The terms "protein," "peptide" and "polypeptide" are used
interchangeably herein, and refer to a polymer of amino acid
residues linked together by peptide (amide) bonds. The terms refer
to a protein, peptide, or polypeptide of any size, structure, or
function. Typically, a protein, peptide, or polypeptide will be at
least three amino acids long. A protein, peptide, or polypeptide
may refer to an individual protein or a collection of proteins. One
or more of the amino acids in a protein, peptide, or polypeptide
may be modified, for example, by the addition of a chemical entity
such as a carbohydrate group, a hydroxyl group, a phosphate group,
a farnesyl group, an isofarnesyl group, a fatty acid group, a
linker for conjugation, functionalization, or other modification,
etc. A protein, peptide, or polypeptide may also be a single
molecule or may be a multi-molecular complex. A protein, peptide,
or polypeptide may be just a fragment of a naturally occurring
protein or peptide. A protein, peptide, or polypeptide may be
naturally occurring, recombinant, or synthetic, or any combination
thereof. In some embodiments a peptide is between 3 and 60 amino
acids long, e.g., between 3 and 15, 15 and 30, 30 and 45, or 45 and
60 amino acids long.
[0068] The term "conjugated" or "conjugation" refers to an
association of two molecules, for example, two proteins or a
protein and a small molecule or other entity, with one another in a
way that they are linked by a direct or indirect covalent or
non-covalent interaction. In the context of conjugation via a
sortase mediated reaction, the conjugation is via a covalent bond
the formation of which is catalyzed by sortase. In the context of
conjugation via click chemistry, the conjugation is via a covalent
bond formed by the reaction of two click chemistry handles. In some
embodiments, a protein is post-translationally conjugated to
another molecule, for example, a second protein, by forming a
covalent bond between the protein and the other molecule after the
protein has been translated, and, in some embodiments, after the
protein has been isolated. In some embodiments, two molecules are
conjugated directly to each other. In some embodiments two
molecules are conjugated via a linker connecting both molecules.
For example, in some embodiments where two proteins are conjugated
to each other to form a protein fusion, the two proteins may be
conjugated via a polypeptide linker, e.g., an amino acid sequence
connecting the C-terminus of one protein to the N-terminus of the
other protein. In some embodiments, a protein N-terminus is
conjugated to or near a C-terminus of a second protein generating
an N--C conjugated chimeric protein. In some embodiments, two
proteins are conjugated at their respective C-termini, generating a
C--C conjugated chimeric protein. In some embodiments, two proteins
are conjugated at their respective N-termini, generating an N--N
conjugated chimeric protein.
[0069] As used herein, a "detectable label" refers to a moiety that
has at least one element, isotope, or functional group incorporated
into the moiety which enables detection of the molecule, e.g., a
protein or polypeptide, or other entity, to which the label is
attached. Labels can be directly attached (i.e., via a bond) or can
be attached by a tether (such as, for example, an optionally
substituted alkylene; an optionally substituted alkenylene; an
optionally substituted alkynylene; an optionally substituted
heteroalkylene; an optionally substituted heteroalkenylene; an
optionally substituted heteroalkynylene; an optionally substituted
arylene; an optionally substituted heteroarylene; or an optionally
substituted acylene, or any combination thereof, which can make up
a tether). It will be appreciated that the label may be attached to
or incorporated into a molecule, for example, a protein,
polypeptide, or other entity, at any position.
[0070] In general, a label can fall into any one (or more) of five
classes: a) a label which contains isotopic moieties, which may be
radioactive or heavy isotopes, including, but not limited to,
.sup.2H, .sup.3H, .sup.13C, .sup.14C, .sup.15N, .sup.18F, .sup.31P,
.sup.32P, .sup.35S, .sup.67Ga, .sup.76Br, .sup.99mTc (Tc-99m),
.sup.111In, .sup.123I, .sup.125I, .sup.131I, .sup.153Gd,
.sup.169Yb, and .sup.186Re; b) a label which contains an immune
moiety, which may be antibodies or antigens, which may be bound to
enzymes (e.g., such as horseradish peroxidase); c) a label which is
a colored, luminescent, phosphorescent, or fluorescent moieties
(e.g., such as the fluorescent label fluoresceinisothiocyanate
(FITC); d) a label which has one or more photo affinity moieties;
and e) a label which is a ligand for one or more known binding
partners (e.g., biotin-streptavidin, FK506-FKBP). In certain
embodiments, a label comprises a radioactive isotope, preferably an
isotope which emits detectable particles, such as .beta. particles.
In certain embodiments, the label comprises a fluorescent moiety.
In certain embodiments, the label is the fluorescent label
fluoresceinisothiocyanate (FITC). In certain embodiments, the label
comprises a ligand moiety with one or more known binding partners.
In certain embodiments, the label comprises biotin. In some
embodiments, a label is a fluorescent polypeptide (e.g., GFP or a
derivative thereof such as enhanced GFP (EGFP)) or a luciferase
(e.g., a firefly, Renilla, or Gaussia luciferase). It will be
appreciated that, in certain embodiments, a label may react with a
suitable substrate (e.g., a luciferin) to generate a detectable
signal. Non-limiting examples of fluorescent proteins include GFP
and derivatives thereof, proteins comprising chromophores that emit
light of different colors such as red, yellow, and cyan fluorescent
proteins, etc. Exemplary fluorescent proteins include, e.g.,
Sirius, Azurite, EBFP2, TagBFP, mTurquoise, ECFP, Cerulean, TagCFP,
mTFP1, mUkG1, mAG1, AcGFP1, TagGFP2, EGFP, mWasabi, EmGFP, TagYPF,
EYFP, Topaz, SYFP2, Venus, Citrine, mKO, mKO2, mOrange, mOrange2,
TagRFP, TagRFP-T, mStrawberry, mRuby, mCherry, mRaspberry, mKate2,
mPlum, mNeptune, mTomato, T-Sapphire, mAmetrine, mKeima. See, e.g.,
Chalfie, M. and Kain, S R (eds.) Green fluorescent protein:
properties, applications, and protocols (Methods of biochemical
analysis, v. 47). Wiley-Interscience, Hoboken, N.J., 2006, and/or
Chudakov, D M, et al., Physiol Rev. 90(3):1103-63, 2010 for
discussion of GFP and numerous other fluorescent or luminescent
proteins. In some embodiments, a label comprises a dark quencher,
e.g., a substance that absorbs excitation energy from a fluorophore
and dissipates the energy as heat.
[0071] The term "adjuvant" encompasses substances that accelerate,
prolong, or enhance the immune response to an antigen. In some
embodiments an adjuvant serves as a lymphoid system activator that
enhances the immune response in a relatively non-specific manner, e
g., without having any specific antigenic effect itself. For
example, in some embodiments an adjuvant stimulates one or more
components of the innate immune system. In certain embodiments an
adjuvant enhances antigen-specific immune responses when used in
combination with a specific antigen or antigens, e.g., as a
component of a vaccine. Adjuvants include, but are not limited to,
aluminum salts (alum) such as aluminum hydroxide or aluminum
phosphate, complete Freund's adjuvant, incomplete Freund's
adjuvant, surface active substances such as lysolecithin, pluronic
polyols, Amphigen, Avridine, bacterial lipopolysaccharides,
3-O-deacylated monophosphoryl lipid A, synthetic lipid A analogs or
aminoalkyl glucosamine phosphate compounds (AGP), or derivatives or
analogs thereof (see, e.g., U.S. Pat. No. 6,113,918),
L121/squalene, muramyl dipeptide, polyanions, peptides, saponins,
oil or hydrocarbon and water emulsions, particles such as ISCOMS
(immunostimulating complexes), etc. In some embodiments an adjuvant
stimulates dendritic cell maturation. In some embodiments an
adjuvant stimulates expression of one or more costimulator(s), such
as B7 or a B7 family member, by antigen presenting cells (APCs),
e.g., dendritic cells. In some embodiments an adjuvant comprises a
CD40 agonist. In some embodiments a CD40 agonist comprises an
anti-CD40 antibody. In some embodiments a CD40 agonist comprises a
CD40 ligand, such as CD40L. In some embodiments an adjuvant
comprises a ligand for a Toll-like receptor (TLR). In some
embodiments an agent is a ligand for one or more of TLRs 1-13,
e.g., at least for TLR3, TLR4, and/or TLR9. In some embodiments an
adjuvant comprises a pathogen-derived molecular pattern (PAMP) or
mimic thereof. In some embodiments an adjuvant comprises an
immunostimulatory nucleic acid, e.g., a double-stranded nucleic
acid, e.g., double-stranded RNA or an analog thereof. For example,
in some embodiments an adjuvant comprises
polyriboinosinic:polyribocytidylic acid (polyIC). In some
embodiments an adjuvant comprises a nucleic acid comprising
unmethylated nucleotides, e.g., a single-stranded CpG
oligonucleotide. In some embodiments an adjuvant comprises a
cationic polymer, e.g., a poly(amino acid) such as poly-L-lysine,
poly-L-arginine, or poly-L-ornithine. In some embodiments an
adjuvant comprises a nucleic acid (e.g., dsRNA, polyIC) and a
cationic polymer. For example, in some embodiments an adjuvant
comprises polyIC and poly-L-lysine. In some embodiments an adjuvant
comprises a complex comprising polyIC, poly-L-lysine, and
carboxymethylcellulose (referred to as polyICLC). In some
embodiments an adjuvant comprises a CD40 agonist and a TLR ligand.
For example, in some embodiments an adjuvant comprises (i) an
anti-CD40 antibody and (ii) an immunostimulatory nucleic acid
and/or a cationic polymer. In some embodiments an adjuvant
comprises an anti-CD40 antibody, an immunostimulatory nucleic acid,
and a cationic polymer. In some embodiments an adjuvant comprises
(i) an anti-CD40 antibody and (ii) poly(IC) or poly(ICLC).
Exemplary adjuvants of use in various embodiments are disclosed in,
e.g., WO/2007/137427 and/or in WO/2009/086640 and/or in one or more
references therein. In certain embodiments an adjuvant is
pharmaceutically acceptable for administration to a human subject.
In certain embodiments an adjuvant is pharmaceutically acceptable
for administration to a non-human subject, e.g., for veterinary
purposes.
[0072] The term "antibody", as used herein, refers to a
glycoprotein belonging to the immunoglobulin superfamily. The terms
antibody and immunoglobulin are used interchangeably. With some
exceptions, mammalian antibodies are typically made of basic
structural units each with two large heavy chains and two small
light chains. There are several different types of antibody heavy
chains, and several different kinds of antibodies, which are
grouped into different isotypes based on which heavy chain they
possess. Five different antibody isotypes are known in mammals,
IgG, IgA, IgE, IgD, and IgM, which perform different roles, and
help direct the appropriate immune response for each different type
of foreign object they encounter. In some embodiments, an antibody
is an IgG antibody, e.g., an antibody of the IgG1, 2, 3, or 4 human
subclass. Antibodies from non-mammalian species (e.g., from birds,
reptiles, amphibia) are also within the scope of the term, e.g.,
IgY antibodies.
[0073] Only part of an antibody is involved in the binding of the
antigen, and antigen-binding antibody fragments, their preparation
and use, are well known to those of skill in the art. As is
well-known in the art, only a small portion of an antibody
molecule, the paratope, is involved in the binding of the antibody
to its epitope (see, in general, Clark, W. R. (1986) The
Experimental Foundations of Modern Immunology Wiley & Sons,
Inc., New York; Roitt, I. (1991) Essential Immunology, 7th Ed.,
Blackwell Scientific Publications, Oxford). The pFc' and Fc
regions, for example, are effectors of the complement cascade but
are not involved in antigen binding. An antibody from which the
pFc' region has been enzymatically cleaved, or which has been
produced without the pFc' region, designated an F(ab') fragment (or
F(ab') 2 fragment), retains both of the antigen binding sites of an
intact antibody. Similarly, an antibody from which the Fc region
has been enzymatically cleaved, or which has been produced without
the Fc region, designated an Fab fragment, retains one of the
antigen binding sites of an intact antibody molecule. Fab fragments
consist of a covalently bound antibody light chain and a portion of
the antibody heavy chain denoted Fd. The Fd fragments are the major
determinant of antibody specificity (a single Fd fragment may be
associated with up to ten different light chains without altering
antibody specificity) and Fd fragments retain epitope-binding
ability in isolation.
[0074] Within the antigen-binding portion of an antibody, as is
well-known in the art, there are complementarity determining
regions (CDRs), which directly interact with the epitope of the
antigen, and framework regions (FRs), which maintain the tertiary
structure of the paratope (see, in general, Clark, W. R. (1986) The
Experimental Foundations of Modern Immunology Wiley & Sons,
Inc., New York; Roitt, I. (1991) Essential Immunology, 7th Ed.,
Blackwell Scientific Publications, Oxford). In both the heavy chain
Fd fragment and the light chain of IgG immunoglobulins, there are
four framework regions (FR1 through FR4) separated respectively by
three complementarity determining regions (CDR1 through CDR3). The
CDRs, and in particular the CDR3 regions, and more particularly the
heavy chain CDR3, are largely responsible for antibody
specificity.
[0075] It is well-established in the art that the non-CDR regions
of a mammalian antibody may be replaced with similar regions of
nonspecific or heterospecific antibodies while retaining the
epitopic specificity of the original antibody. This is most clearly
manifested in the development and use of "humanized" antibodies in
which non-human CDRs are covalently joined to human FR and/or
Fc/pFc' regions to produce a functional antibody. See, e.g., U.S.
Pat. Nos. 4,816,567, 5,225,539, 5,585,089, 5,693,762, and
5,859,205.
[0076] Fully human monoclonal antibodies also can be prepared by
immunizing mice transgenic for large portions of human
immunoglobulin heavy and light chain loci. Following immunization
of these mice (e.g., XenoMouse (Abgenix), HuMAb mice
(Medarex/GenPharm)), monoclonal antibodies can be prepared
according to standard hybridoma technology. These monoclonal
antibodies will have human immunoglobulin amino acid sequences and
therefore will not provoke human anti-mouse antibody (HAMA)
responses when administered to humans.
[0077] Thus, as will be apparent to one of ordinary skill in the
art, the present invention also provides for F(ab'), Fab, Fv, and
Fd fragments; antibodies in which the Fc and/or FR and/or CDR1
and/or CDR2 and/or light chain CDR3 regions have been replaced by
homologous human or non-human sequences; antibodies in which the FR
and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been
replaced by homologous human or non-human sequences; antibodies in
which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3
regions have been replaced by homologous human or non-human
sequences; and antibodies in which the FR and/or CDR1 and/or CDR2
regions have been replaced by homologous human or non-human
sequences. In some embodiments, the present invention provides for
so-called single chain antibodies (e.g., scFv), (single) domain
antibodies (sdAb), and other antibodies, which, in some
embodiments, find use as intracellular antibodies. A single-chain
variable fragment (scFv) is a protein comprising the variable
regions of the heavy (VH) and light chains (VL) of an
immunoglobulin, connected with a short linker peptide of, e.g.,
about 10 to about 25 amino acids. A divalent (or bivalent)
single-chain variable fragment (di-scFvs, bi-scFvs) can be
engineered by linking two scFvs, e.g., by producing a single
peptide chain with two VH and two VL regions, yielding tandem
scFvs. Two sdAbs or an sdAb and an scFv can also be linked by
producing them as single polypeptide chains. In some embodiments
two scFv are joined in the form of a diabody. By using a linker
that is too short to allow pairing between the two domains (VH and
VL) on the same scFv chain (e.g., less than about 10 amino acids,
e.g., about 5 amino acids) the domains instead pair with the
complementary domains of another scFv chain and thereby create two
antigen-binding sites. A bispecific agent may be created by linking
the VH and VL of two different antibodies A and B to form two
different "cross-over" chains VH.sub.A-VL.sub.B and
VH.sub.B-VL.sub.A, whereby the chains recreate both antigen-binding
sites on association (see, e.g., P., et al., Proc Natl Acad Sci
USA. 1993; 90(14):6444-8). Domain antibodies, camelid and camelized
antibodies and fragments thereof, for example, VHH domains, or
nanobodies, such as those described in patents and published patent
applications of Ablynx NV and Domantis are also encompassed in the
term antibody. Also encompassed are VH domains obtained or derived
from immunoglobulin novel (or new) antigen receptors (IgNAR) found
in cartilaginous fish (e.g., sharks, skates and rays). See, e.g.,
WO 05/18629; Barelle, C., et al., Adv Exp Med Biol. (2009)
655:49-62, and/or the chapter by Flajnik and Dooley in Antibody
Phage Display: Methods and Protocols, Methods in Molecular Biology,
2009. The term "antigen-binding antibody fragment," as used herein,
refers to a fragment of an antibody that comprises the paratope, or
a fragment of the antibody that binds to the antigen to which the
antibody binds, with similar specificity and affinity as the intact
antibody. Where the present disclosure refers to antibodies, the
disclosure provides embodiments pertaining to or using
antigen-binding fragments of such antibodies.
[0078] Antibodies, e.g., fully human monoclonal antibodies, may be
identified using phage display (or other display methods such as
yeast display, ribosome display, bacterial display). Display
libraries, e.g., phage display libraries, are available (and/or can
be generated by one of ordinary skill in the art) that can be
screened to identify an antibody that binds to an antigen of
interest, e.g., using panning. See, e.g., Sidhu, S. (ed.) Phage
Display in Biotechnology and Drug Discovery (Drug Discovery Series;
CRC Press; 1.sup.st ed., 2005; Aitken, R. (ed.) Antibody Phage
Display: Methods and Protocols (Methods in Molecular Biology)
Humana Press; 2nd ed., 2009. In some embodiments, a monoclonal
antibody is produced using recombinant methods in suitable host
cells, e.g., prokaryotic or eukaryotic host cells. In some
embodiments microbial host cells (e.g., bacteria, fungi) are used.
Nucleic acids encoding antibodies or portions thereof may be
isolated and their sequence determined. Such nucleic acid sequences
may be inserted into suitable vectors (e.g., plasmids) and, e.g.,
introduced into host cells for expression. In some embodiments
insect cells are used. In some embodiments mammalian cells, e.g.,
human cells, are used. In some embodiments, an antibody is secreted
by host cells that produce it and may be isolated, e.g., from
culture medium. Methods for production and purification of
recombinant proteins are well known to those of ordinary skill in
the art. It will be understood that such methods may be applied to
produce and, optionally, purify, any protein of interest
herein.
[0079] The term "chimeric antigen receptor" (CAR) refers to a
polypeptide comprising a cell activation domain fused to a binding
domain, e.g., a domain comprising an antigen binding moiety. A cell
that expresses a chimeric antigen receptor may be referred to as a
"CAR cell". In general, the binding domain is or is located in an
extracellular domain of the polypeptide, and the activation domain
is located inside the cell in the cytoplasm (cytoplasmic domain).
Upon binding of binding moiety to a ligand, the activation domain
transmits an activation signal to the CAR cell, and the cell
becomes activated as a result of signaling via the activation
domain. For example, upon binding of the antigen binding moiety to
its cognate antigen (e.g., a protein, lipid, or other molecule) the
activation domain transmits an activation signal to the CAR cell,
and the cell becomes activated as a result of signaling via the
activation domain. If the antigen is expressed by a target cell,
the antigen binding moiety directs the specificity of the CAR cell
to a target cell of interest. Effector functions of the CAR cell,
such as cell-mediated cytotoxicity, are directed to the target
cell. In some embodiments the CAR cell is a T cell. In some
embodiments the CAR cell is an NK cell. In some embodiments the
binding domain, e.g., antigen binding domain, comprises a single
chain variable fragment. The binding domain is typically preceded
by a signal peptide to direct the nascent CAR to the endoplasmic
reticulum and subsequent surface expression. In general, any
eukaryotic signal peptide sequence may be used. In some embodiments
the signal peptide natively attached to the amino-terminal most
component of the CAR is used. It will be appreciated that the
signal peptide is cleaved and therefore absent in the mature CAR.
In some embodiments the cell activation domain comprises a
biologically active portion of the signaling domain of an antigen
receptor such as the T cell receptor complex (TCR-CD3 complex). For
example, in some embodiments the cell activation domain comprises
at least a portion of the cytoplasmic domain (endodomain) of the T
cell receptor CD3.zeta. (zeta) chain (CD247) or CD3.epsilon.
(epsilon) chain that is sufficient to activate T cells. In some
embodiments a CAR comprises all or substantially all of the
cytoplasmic domain of the CD3.zeta. chain. In some embodiments, at
least a portion of CD3.zeta. comprising 1, 2, or 3 ITAM motifs is
used. A CAR typically comprises a transmembrane domain (TMD)
between the extracellular and cytoplasmic domains. In general, a
TMD may comprise at least a portion of a TMD found in any
transmembrane protein, e.g., any human transmembrane protein. In
some embodiments the transmembrane protein is a protein that spans
the plasma membrane. In some embodiments the sequence of a TMD of a
CAR comprises at least the sequence of an alpha helical region of a
naturally occurring transmembrane protein. In some embodiments a
TMD is derived from the alpha or beta chain of the T-cell receptor,
CD28, CD3 zeta, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22,
CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. In some
embodiments a synthetic TMD may be used. One of ordinary skill in
the art will be aware of numerous transmembrane proteins and can
readily select a TMD or design a synthetic TMD (see, e.g., Sharpe,
H J, et al, Cell. 2010l 142(1):158-69 for discussion of features of
TMDs and numerous examples of such domains (e.g., in Table S2)). In
some embodiments a CAR comprises the CD3zeta transmembrane domain
and endodomain. In some embodiments a CAR comprises a spacer region
one or more amino acids long (e.g., a polypeptide linker) that
links the binding domain to the transmembrane domain. A spacer
region sufficiently flexible to allow the binding domain to orient
in different directions to facilitate antigen recognition may be
selected. Exemplary spacer regions may comprise, e.g., the hinge
region from an immunoglobulin, e.g., from IgG1, the C.sub.H2
C.sub.H3 region of an immunoglobulin, or portions of CD3. In some
embodiments the cytoplasmic domain of a CAR comprises an activation
domain and at least one domain that provides co-stimulatory
signals. Examples of proteins containing such co-stimulatory
domains include CD28, 4-1BB, DAP10, ICOS, OX40, CD30, CD40,
lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT,
NKNKG2C, B7-H3, a ligand that specifically binds with CD83, and any
combination thereof) to the cytoplasmic portion of the CAR to
provide additional signals to the T cell. In some embodiments a CAR
comprises multiple co-stimulatory domains, which may increase
potency and/or persistence/proliferation of CAR cells that express
the CAR, resulting in designs such as CD3zeta-CD28-4-1BB or
CD3zeta-CD28-OX40 (wherein in each case the name of the molecule
indicates the protein in which the particular signaling domain is
found in nature). The order of such domains described herein is not
to be considered limiting. In some embodiments the transmembrane
domain of the CAR is the transmembrane domain of the most membrane
proximal component of the cytoplasmic domain of the CAR. For
example, if the cytoplasmic domain of the CAR comprises
CD3zeta-CD28-4-1BB, with the CD3zeta component being located
closest to the inner face of the plasma membrane (i.e., separated
from the inner face of the plasma membrane by the fewest amino
acids), the CAR comprises the transmembrane domain of CD3zeta. It
should be understood that any of the domains of a CAR may be
connected via a spacer rather than being directly fused to one
another. Nucleic acid constructs encoding CARs may be introduced
into cells by any suitable method, e.g., by lentiviral or
gamma-retroviral vector gene transfer or by electroporation.
[0080] Examples of CARs, CAR cells, methods of making, culturing,
manipulating, storing, and using CARs and CAR cells, reagents
useful for generating CAR cells such as nucleic acid constructs and
vectors encoding CARs, are described in the following publications:
U.S. Pat. Pub. Nos: 20040038886, 20110158957; 20120148552,
20130071414, 20130266551, 20130280285; 20130287748; PCT application
publications WO/2012/079000 (PCT/US11/064191) and/or Finney, H M,
et al., J Immunol. 2004; 172(1):104-13; Kalos, M., et al., Sci.
Transl. Med. 3, 95ra73 (2011); Porter, D L, et al., N Engl J Med
2011; 365:725-33. Such CARs, CAR cells, methods of making,
culturing, manipulating, storing, and using CARs and CAR cells,
reagents useful for generating CAR cells such as nucleic acid
constructs and vectors encoding CARs, may be used in certain
embodiments of the present invention. Furthermore, methods,
compositions reagents, media, or devices described in any of the
foregoing references may, wherever applicable, be used in
embodiments of the present invention that pertain to CAR cells or
that do not pertain to CAR cells. Such methods, reagents, media, or
devices may, for example, pertain to obtaining, culturing,
maintaining, manipulating, expanding, storing, and/or administering
cells. In some aspects, methods described herein of sortagging
cells, wherein an agent is conjugated to an endogenous,
non-genetically engineered polypeptide expressed by a cell, may be
applied to CAR cells described in or generated as described in any
of the afore-mentioned references. In some embodiments, sortagged
CAR cells may be used to treat a cancer that contains cells that
express an antigen to which the CAR binds or to treat an infection
in which infected cells express an antigen to which the CAR binds.
In some embodiments, any CAR that comprises an antigen binding
moiety that binds to a particular antigen or epitope may be
modified by replacing the antigen binding moiety with a different
antigen binding moiety that binds to the same antigen or epitope.
In some embodiments, any CAR that comprises an antigen-binding
moiety that binds to a particular antigen may be modified by
replacing the antigen-binding moiety with an antigen-binding moiety
that binds to a different antigen, to generate a CAR that binds to
such different antigen or epitope. Such modification may be
accomplished by modifying the nucleic acid construct used to
produce the CAR, e.g., by replacing the sequence that encodes the
antigen-binding moiety with a sequence that encodes a different
antigen-binding moiety. CAR T cells and CAR NK cells that express
CARs that specifically bind to various tumor antigens (e.g., CD19,
CD20, etc.) have been tested in clinical trials for the treatment
of human subjects with cancer (e.g., hematologic malignancies) and
shown benefit in some patients. The present invention contemplates
the sortagging of any CAR cells that have shown reasonable safety
in a clinical trial.
[0081] The term "chimeric antibody," as used herein, refers to an
antibody, or an antigen-binding antibody fragment, conjugated to
another molecule, for example, to a second antibody, or
antigen-binding antibody fragment. Any antibody or antigen-binding
antibody fragment, or antigen-binding protein domain can be used to
generate a chimeric antibody. In some embodiments, a chimeric
antibody comprises two conjugated antibodies, or antibody
fragments, or one antibody conjugated to an antibody fragment,
wherein the antigen-binding domains of the conjugated molecules
bind different antigens or different epitopes of the same antigen.
Such chimeric antibodies are referred to herein as "bi-specific,"
since they bind two different antigens/epitopes.
[0082] The term "costimulator" refers to a molecule that provides a
stimulus (or second signal) that promotes or is required, in
addition to antigen, for stimulation of naive immune system cells,
e.g., naive T cells or naive B cells, and/or that contributes to
sustaining or modifying the response. Naturally occurring
costimulators include various molecules expressed on the surface of
or secreted by APCs, which molecules bind to receptors on the
surfaces of, e.g., T cells. Examples of receptors to which
costimulators bind include, e.g., CD28 family members (e.g., CD28
and inducible costimulator (ICOS)) and CD2 family members (e.g.,
CD2, SLAM). Examples of costimulators include various members of
the B7 family of molecules such as B7-1 and B7-2 (which bind to
CD28) and ICOS ligand (which binds to ICOS). In some embodiments a
costimulator is a TNF alpha family member. For example, CD70 on DCs
binding to its receptor CD27 on naive T cells delivers
costimulatory signals; 4-1BBL (also called CD137L) on APCs delivers
costimulatory signals by binding to its receptor CD137 on T cells.
It will be appreciated that the effects of an interaction may be
bidirectional, e.g., APCs may receive costimulation via their
interaction with cells that they stimulate. OX40 (CD134) is a
secondary costimulatory molecule, expressed after typically about
24 to 72 hours following activation; its ligand, OX40L, is
expressed on APCs following their activation. In some embodiments
expression of costimulator(s) by APCs is stimulated by an adjuvant,
e.g., a CD40 ligand, PAMP or PAMP mimic, or TLR ligand. In some
embodiments a costimulator is a soluble molecule. In some
embodiments a soluble costimulator is a recombinantly produced
polypeptide comprising at least a functional portion of the
extracellular domain of a naturally occurring costimulator or a
functional variant thereof.
[0083] The term "endogenous polypeptide" refers to a naturally
occurring polypeptide that originates naturally from or is
naturally produced by a cell, e.g., a polypeptide that is an
expression product of a gene that both (i) is present in the
genetic material (nuclear or mitochondrial genome) of the cell (an
"endogenous gene") and (ii) has not been modified or introduced
into the cell or an ancestor of the cell by the hand of man or by a
virus or other vector. It will be understood that endogenous genes
of a particular species (e.g., humans) may include sequences
introduced by retroviruses, transposons, or other vectors but that
have been present in the genome of at least some members of the
species for sufficiently long to be considered endogenous. For
purposes hereof, genetic elements that can be shown to have been
present in the genome of at least some individuals of a particular
species, e.g., at a particular chromosomal location, for at least
1000 years are considered endogenous to that species. One of
ordinary skill in the art will be aware of endogenous genes and
polypeptides of animal cells, e.g., mammalian cells, e.g., human
cells. For purposes hereof, "introducing" a nucleic acid into a
cell encompasses introducing the nucleic acid itself or introducing
a nucleic acid that can undergo one or more rounds of copying,
reverse transcription, and/or processing in the cell to yield the
nucleic acid. An endogenous polypeptide may be processed or
modified during or after its synthesis. For example an N-terminal
amino acid or secretion signal sequence may be removed or a loop
may be cleaved. In certain embodiments of any aspect of the
disclosure, "endogenous polypeptides" are also not chemically
modified as defined below.
[0084] The terms "genetically engineered" or "genetically
modified", or "recombinant" encompass nucleic acids whose sequence
comprises a non-naturally occurring sequence, (a sequence invented
or generated by man and not occurring in nature or not known to
occur in nature), comprises two or more naturally occurring
sequences joined together that are not found joined to one another
in their naturally occurring state, or comprises a deletion,
insertion, rearrangement, or other alteration of or in a naturally
occurring sequence, wherein the deletion, insertion, rearrangement,
or other alteration is brought about by the hand of man. The terms
"genetically engineered", "genetically modified", or "recombinant"
polypeptide encompass polypeptides encoded by genetically
engineered nucleic acids. In some embodiments the sequence of a
genetically engineered polypeptide expressed by a cell is distinct
from those polypeptides that are endogenous to the cell. The terms
"genetically engineered cell", "genetically modified cell", or
"recombinant" cell" encompass cells into which a nucleic acid has
been introduced by the hand of man and their descendants that
inherit at least a portion of the introduced nucleic acid. In some
embodiments a genetically engineered cell has had its genome
altered by the hand of man, e.g., by insertion of an exogenous
nucleic acid sequence and/or deletion of an endogenous nucleic acid
sequence, or is descended from such a cell and has inherited a copy
of at least a portion of the original alteration. In some
embodiments the nucleic acid or a portion thereof, or a copy of the
nucleic acid or a portion thereof, may be integrated into the
genome of the cell. "Non-genetically engineered, "non-genetically
modified", and "non-recombinant" refer to not being genetically
engineered, absence of genetic modification, etc. Non-genetically
engineered polypeptides encompass endogenous polypeptides. In
certain embodiments a non-genetically engineered cell, gene, or
genome does not contain non-endogenous nucleic acid, e.g., DNA or
RNA that originates from a vector, from a different species, or
that comprises an artificial sequence, e.g., DNA or RNA that was
introduced by the hand of man. In certain embodiments a
non-genetically engineered cell has not been intentionally
contacted with a nucleic acid that is capable of causing a
heritable genetic alteration under conditions suitable for uptake
of the nucleic acid by the cells.
[0085] The terms "chemically engineered" or "chemically modified"
encompass modifications made to endogenous proteins or cells to
introduce a "linker" as described below, to an endogenous protein
or cell. Chemical modifications can be any known in the art.
Examples of such modifications are provided in Ta et al. Circ. Res.
2011; 109: 365-373 and International Publication No. WO
2012/142659, both incorporated by reference herein in their
entireties. Other chemical modifications that can be made to
biomolecules, e.g., polypeptides, are known to those of ordinary
skill in the art. See, e.g., Hermanson, G., Bioconjugate
Techniques, Academic Press; 2nd edition (2008). In certain
embodiments, chemical modification of cells includes introducing a
reactive functional group, such as a sulfhydryl or maleimide, to
cell surfaces, e.g., by attachment (e.g., via a covalent bond) to
an extracellular domain of an endogenous polypeptide, followed by
labelling the cells with a moiety capable of serving as a
nucleophile in a sortase-catalyzed reaction, such as a (G).sub.n-
or (A).sub.n-containing peptide, via reaction with such reactive
functional group. In certain embodiments, chemical modification of
cells includes introducing sulfhydryls to cell surfaces via
reaction with primary amines using, e.g., 2-Iminothiolane or
Traut's reagent followed by labelling cells with NH.sub.2GGG-tags
via specific reaction of sulfhydryls on the cell surface and
maleimide groups on NH.sub.2-GGG-maleimide peptides.
[0086] The term "immunomodulator" refers to substances that are
capable either by themselves or together with other agent(s) of
inducing, enhancing, suppressing, or regulating an immune response.
(It will be understood that the term generally does not refer to
those entities that are the target of the immune response such as
pathogens, tumor cells, grafts, or self antigens in the case of
autoimmune disease). Immunomodulators include substances capable of
modulating the activation, proliferation, differentiation, and/or
biological activity of immune system cells. Examples, include,
e.g., cytokines, costimulators, adjuvants.
[0087] The term "linker" as used herein, refers to a chemical group
or molecule covalently linked to a molecule, for example, a
protein, and a chemical group or moiety. In some embodiments, the
linker is positioned between, or flanked by, two groups, molecules,
or moieties and connected to each one via a covalent bond, thus
connecting the two. In some embodiments, the linker is an amino
acid or a plurality of amino acids. In some embodiments, the linker
is an organic molecule, group, or chemical moiety. In some
embodiments a linker connects two or more polypeptides. In some
embodiments a linker comprises or consists of a polypeptide. In
some embodiments a linker may comprise or consist of one or more
glycine residues and, in some embodiments, one or more serine,
and/or threonine residues. In some embodiments, the linker
comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, or more than 20 amino acids. In some embodiments,
the linker comprises an oligoglycine sequence. In some embodiments
a linker may comprise at least 50% glycine residues and, in some
embodiments, between 5% and 50% of the amino acids are serine or
threonine (i.e., S+T is between 5% and 50%, where S is the
percentage of serine residues and T is the percentage of threonine
residues). Examples of linkers include, e.g., (Gly-Ser).sub.n;
(Gly-Gly-Ser).sub.n; (Gly-Gly-Gly-Ser).sub.n;
(Gly-Gly-Gly-Gly-Ser).sub.n, where n is a number sufficient to
produce a desired linker length, e.g., about 5-15 amino acids,
e.g., up to about 25-50 amino acids. The afore-mentioned sequences
can be permuted and/or concatenated in any order and/or may be
truncated and/or any of the Ser residues may be replaced by Thr
and/or any of the Gly or Ser residues may be replaced by Ala. In
some embodiments a linker comprises an aliphatic, alicyclic,
heteroaliphatic, heteroalicyclic, aromatic, or heteroaromatic
linker which, in some embodiments, comprises between 1 and 6, 6 and
12, or 12-30 carbon atoms in the main chain connecting the moieties
at each end. In some embodiments a linker comprises a linear
saturated or unsaturated hydrocarbon chain, an oligo(ethylene
glycol) chain, one or more amino acids (e.g., a peptide), an
alicyclic structure, or an aromatic ring. In some embodiments a
linker is sufficiently long and flexible so as to permit linked
polypeptides to assemble to form a proper three dimensional
structure (e.g., as found in nature) and/or retain one or more
activities such as binding, enzymatic activity, and/or appropriate
interaction with its typical interaction partners. In some
embodiments a linker may comprise a protease recognition site or
labile bond, which allows release of one or both of the linked
moieties under appropriate conditions, e.g., in the presence of a
protease that recognizes the protease recognition site and cleaves
within or near the linker.
[0088] The term "marker" or "cellular marker" refers to any
molecular moiety (e.g., protein, peptide, carbohydrate,
polysaccharide, nucleic acid (mRNA or other RNA species, DNA),
lipid, or a combination thereof) that characterizes, indicates, or
identifies one or more cell type(s), tissue type(s), cell lineages,
or embryological tissue of origin and/or that characterizes,
indicates, or identifies a particular physiological or pathological
state, e.g., an activation state, cell cycle state, metabolic
state, differentiation state, apoptotic state, diseased state, etc.
In some embodiments, the presence, absence, or amount of certain
marker(s) may indicate a particular physiological or diseased state
of a subject, organ, tissue, or cell. In some embodiments a cell
surface marker is a "cluster of differentiation" (CD) molecule.
Numerous CD molecules are known in the art. See, e.g., H. Zola, et
al., Leukocyte and Stromal Cell Molecules: the CD Markers, Wiley,
New Jersey, 2007 and/or databases cited therein; Proceedings of the
9th International Workshop on Human Leukocyte Differentiation
Antigens published in Immunology Letters, Volume 134, Issue 2,
Pages 103-188 (30 Jan. 2011); Human Cell Differentiation Molecules
database available at
http://www.hcdm.org/MoleculeInformation/tabid/54/Default.aspx;
and/or Human and Mouse CD Handbook, available at
http://www.bdbiosciences.com/documents/cd_marker_handbook.pdf (BD
Biosciences, San Jose, Calif., 2010). In some embodiments a
cellular marker is cell type specific. For example, a cell type
specific marker is typically present at a higher level on or in a
particular cell type or cell types of interest than on or in many
other cell types. In some instances a cell type specific marker is
present at detectable levels only on or in a particular cell type
of interest. However, it will be appreciated that useful cell type
specific markers need not be absolutely specific for the cell type
of interest. In some embodiments a cell type specific marker for a
particular cell type is expressed at levels at least 3 fold greater
in that cell type than in a reference population of cells which may
consist, for example, of a mixture containing cells from a
plurality (e.g., 5-10 or more) of different tissues or organs in
approximately equal amounts. In some embodiments a cell type
specific marker is present at levels at least 4-5 fold, between
5-10 fold, or more than 10-fold greater than its average expression
in a reference population. In some embodiments detection or
measurement of a cell type specific marker can distinguish the cell
type or types of interest from cells of many, most, or all other
types. In general, the presence and/or abundance of most markers
may be determined using standard techniques such as Northern
blotting, in situ hybridization, RT-PCR, sequencing, immunological
methods such as immunoblotting, immunodetection, or fluorescence
detection following staining with fluorescently labeled antibodies,
oligonucleotide or cDNA microarray or membrane array, protein
microarray analysis, mass spectrometry, etc.
[0089] The term "naturally occurring" as applied to an entity
(e.g., a molecule, substance, etc.) refers to the fact that the
entity can be found in nature as distinct from being artificially
created or modified by man. For example, a polypeptide or
polynucleotide sequence that is naturally present in a virus or in
a prokaryotic (bacteria) or eukaryotic (e.g., fungal, protozoa,
insect, plant, vertebrate) cell, tissue, or organism and/or that
can be isolated from a source in nature and which has not been
intentionally modified by man (e.g., in the laboratory) is
naturally occurring. "Non-naturally occurring" (also referred to as
"synthetic" or "artificial") as applied to an entity means that the
entity is not naturally occurring, i.e., it cannot be found in
nature as distinct from being artificially produced by man. It will
be appreciated that a "naturally occurring" entity may be produced
by man, e.g., through recombinant nucleic acid techniques or
chemical synthesis and/or may be isolated or purified. Such an
entity is still considered naturally occurring so long as it does
not otherwise differ materially from the entity as found in
nature.
[0090] The term "purified" refers to agents that have been
separated from some, many, or most of the components with which
they are associated in nature or when originally generated. In
general, such purification involves action of the hand of man. In
some embodiments a purified agent is, for example, at least 50%,
60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more than
99% pure. In some embodiments, a nucleic acid, polypeptide, or
small molecule is purified such that it constitutes at least 75%,
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.95%, or
more, of the total nucleic acid, polypeptide, or small molecule
material, respectively, present in a preparation. In some
embodiments, an organic substance, e.g., a nucleic acid,
polypeptide, or small molecule, is purified such that it
constitutes at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%,
99.5%, 99.9%, 99.95%, or more, of the total organic material
present in a preparation. Purity may be based on, e.g., dry weight,
size of peaks on a chromatography tracing (GC, HPLC, etc.),
molecular abundance, electrophoretic methods, intensity of bands on
a gel, spectroscopic data (e.g., NMR), elemental analysis, high
throughput sequencing, mass spectrometry, or any art-accepted
quantification method. In some embodiments, water, buffer
substances, ions, and/or small molecules (e.g., synthetic
precursors such as nucleotides or amino acids), can optionally be
present in a purified preparation. A purified agent may be prepared
by separating it from other substances (e.g., other cellular
materials), or by producing it in such a manner to achieve a
desired degree of purity. In some embodiments "partially purified"
or "at least partially purified" with respect to a molecule
produced by a cell means that a molecule produced by a cell is no
longer present within the cell, e.g., the cell has been lysed and,
optionally, at least some of the cellular material (e.g., cell
wall, cell membrane(s), cell organelle(s)) has been removed and/or
the molecule has been separated or segregated from at least some
molecules of the same type (protein, RNA, DNA, etc.) that were
present in the lysate or, in the case of a molecule that is
secreted by a cell, the molecule has been separated from at least
some components of the medium or environment into which it was
secreted. In some embodiments, any agent disclosed herein is
purified. In some embodiments a composition comprises one or more
purified agents.
[0091] The term "RNA interference" (RNAi) encompasses processes in
which a molecular complex known as an RNA-induced silencing complex
(RISC) silences or "knocks down" gene expression in a
sequence-specific manner in, e.g., eukaryotic cells, e.g.,
vertebrate cells, or in an appropriate in vitro system. RISC may
incorporate a short nucleic acid strand (e.g., about 16-about 30
nucleotides (nt) in length) that pairs with and directs or "guides"
sequence-specific degradation or translational repression of RNA
(e.g., mRNA) to which the strand has complementarity. The short
nucleic acid strand may be referred to as a "guide strand" or
"antisense strand". An RNA strand to which the guide strand has
complementarity may be referred to as a "target RNA". The
complementarity of the structure formed by hybridization of a
target RNA and the guide strand may be such that the strand can (i)
guide cleavage of the target RNA in the RNA-induced silencing
complex (RISC) and/or (ii) cause translational repression of the
target RNA. Reduction of expression due to RNAi may be essentially
complete (e.g., the amount of a gene product is reduced to
background levels) or may be less than complete in various
embodiments. For example, mRNA and/or protein level may be reduced
by 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more, in various
embodiments. As known in the art, the complementarity between the
guide strand and a target RNA need not be perfect (100%) but need
only be sufficient to result in inhibition of gene expression. For
example, in some embodiments 1, 2, 3, 4, 5, or more nucleotides of
a guide strand may not be matched to a target RNA. In some
embodiments a guide strand has at least about 80%, 85%, or 90%,
e.g., least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or
100% sequence complementarity to a target RNA over a continuous
stretch of at least about 15 nt, e.g., between 15 nt and 30 nt,
between 17 nt and 29 nt, between 18 nt and 25 nt, between 19 nt and
23 nt, of the target RNA. In some embodiments at least the seed
region of a guide strand (the nucleotides in positions 2-7 or 2-8
of the guide strand) is perfectly complementary to a target RNA. As
used herein, the term "RNAi agent" encompasses nucleic acids that
can be used to achieve RNAi in eukaryotic cells. Short interfering
RNA (siRNA), short hairpin RNA (shRNA), and microRNA (miRNA) are
examples of RNAi agents. siRNAs typically comprise two separate
nucleic acid strands that are hybridized to each other to form a
structure that contains a double stranded (duplex) portion at least
15 nt in length, e.g., about 15-about 30 nt long, e.g., between
17-27 nt long, e.g., between 18-25 nt long, e.g., between 19-23 nt
long, e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, or 30 nucleotides. In some embodiments the strands of an siRNA
are perfectly complementary to each other within the duplex
portion. In some embodiments the duplex portion may contain one or
more unmatched nucleotides, e.g., one or more mismatched
(non-complementary) nucleotide pairs or bulged nucleotides. In some
embodiments either or both strands of an siRNA may contain up to
about 1, 2, 3, or 4 unmatched nucleotides within the duplex
portion. In some embodiments a strand may have a length of between
15-35 nt, e.g., between 17-29 nt, e.g., 19-25 nt, e.g., 21-23 nt.
Strands may be equal in length or may have different lengths in
various embodiments. In some embodiments strands may differ by
between 1-10 nt in length. A strand may have a 5' phosphate group
and/or a 3' hydroxyl (--OH) group. Either or both strands of an
siRNA may comprise a 3' overhang of, e.g., about 1-10 nt (e.g., 1-5
nt, e.g., 2 nt). shRNAs are nucleic acid molecules that comprise a
stem-loop structure and a length typically between about 40-150 nt,
e.g., about 50-100 nt, e.g., 60-80 nt. A "stem-loop structure"
(also referred to as a "hairpin" structure) refers to a nucleic
acid having a secondary structure that includes a region of
nucleotides which are known or predicted to form a double strand
(stem portion; duplex) that is linked on one side by a region of
(usually) predominantly single-stranded nucleotides (loop portion).
Such structures are well known in the art and the term is used
consistently with its meaning in the art. A guide strand sequence
may be positioned in either arm of the stem, i.e., 5' with respect
to the loop or 3' with respect to the loop in various embodiments.
As is known in the art, the stem structure does not require exact
base-pairing (perfect complementarity). Thus, the stem may include
one or more unmatched residues or the base-pairing may be exact,
i.e., it may not include any mismatches or bulges. In some
embodiments the stem is between 15-30 nt, e.g., between 17-29 nt,
e.g., 19-25 nt. In some embodiments the stem is between 15-19 nt.
In some embodiments the stem is between 19-30 nt. In some
embodiments the loop is between 1 and 20 nt in length, e.g., 1-15
nt, e.g., 4-9 nt. The shRNA structure may comprise a 5' or 3'
overhang. As known in the art, an shRNA may undergo intracellular
processing to remove the loop and generate an siRNA. Mature
endogenous miRNAs are short (typically 18-24 nt, e.g., about 22
nt), single-stranded RNAs that are generated by intracellular
processing from larger, endogenously encoded precursor RNA
molecules termed miRNA precursors (see, e.g., Bartel, D., Cell.
116(2):281-97 (2004); Bartel D P. Cell. 136(2):215-33 (2009);
Winter, J., et al., Nature Cell Biology 11: 228-234 (2009).
Artificial miRNA may be designed to take advantage of the
endogenous RNAi pathway in order to silence a target RNA of
interest. An RNAi agent that contains a strand sufficiently
complementary to an RNA of interest so as to result in reduced
expression of the RNA of interest (e.g., as a result of degradation
or repression of translation of the RNA) in a cell or in an in
vitro system capable of mediating RNAi and/or that comprises a
sequence that is at least 80%, 90%, 95%, or more (e.g., 100%)
complementary to a sequence comprising at least 10, 12, 15, 17, or
19 consecutive nucleotides of an RNA of interest may be referred to
as being "targeted to" the RNA of interest. An RNAi agent targeted
to an RNA transcript may also considered to be targeted to a gene
from which the transcript is transcribed. In some embodiments an
RNAi agent is a vector (e.g., an expression vector) suitable for
causing intracellular expression of one or more transcripts that
give rise to a siRNA, shRNA, or miRNA in the cell. Such a vector
may be referred to as an "RNAi vector". An RNAi vector may comprise
a template that, when transcribed, yields transcripts that may form
a siRNA (e.g., as two separate strands that hybridize to each
other), shRNA, or miRNA precursor (e.g., pri-miRNA or
pre-mRNA).
[0092] The term "sortagging," as used herein, refers to the process
of attaching (conjugating) a tag to a target entity, e.g., a
molecule, for example, a protein, using a sortase. The term
"sortagging" encompasses attaching a tag to a protein expressed by
a living cell using a sortase, thereby attaching the tag to the
cell. The term "tag" is used in a broad sense to encompass any of a
wide variety of entities. Examples of suitable tags include, but
are not limited to, amino acids, peptides, proteins, nucleic acids,
polynucleotides, sugars, carbohydrates, polymers, lipids, fatty
acids, and small molecules. Other suitable tags will be apparent to
those of skill in the art and the invention is not limited in this
aspect. In some embodiments a tag is covalently or noncovalently
attached to, physically associated with, or part of another entity,
such as a virus, cell, particle, or other supramolecular complex,
and attaching the tag to the target entity (sortagging the target
entity) attaches the entity to the target entity. In some
embodiments, a tag comprises a sequence useful for purifying,
expressing, solubilizing, and/or detecting a polypeptide. In some
embodiments, a tag may comprise two or more moieties, which may be
conjugated to each other. In some embodiments a tag may serve
multiple functions. In some embodiments a tag is a relatively small
polypeptide, e.g., ranging from a few amino acids up to about 100
amino acids long. In some embodiments a tag is more than 100 amino
acids long, e.g., up to about 500 amino acids long, or more. In
some embodiments, a tag comprises an HA, TAP, Myc, 6.times.His,
Flag, V5, or GST tag, to name few examples. A tag (e.g., any of the
afore-mentioned tags) that comprises an epitope against which an
antibody, e.g., a monoclonal antibody, is available (e.g.,
commercially available) or known in the art may be referred to as
an "epitope tag". In some embodiments a tag comprises a
solubility-enhancing tag (e.g., a SUMO tag, NUS A tag, SNUT tag, a
Strep tag, or a monomeric mutant of the Ocr protein of
bacteriophage T7). See, e.g., Esposito D and Chatterjee D K. Curr
Opin Biotechnol.; 17(4):353-8 (2006). In some embodiments, a tag is
cleavable, so that at least a portion of it can be removed, e.g.,
by a protease. In some embodiments, this is achieved by including a
protease cleavage site in the tag, e.g., adjacent or linked to a
functional portion of the tag. Exemplary proteases include, e.g.,
thrombin, TEV protease, Factor Xa, PreScission protease, etc. In
some embodiments, a "self-cleaving" tag is used. See, e.g.,
PCT/US05/05763. In some embodiments, sortagging involves coupling a
tag to an endogenous protein on the surface of a cell.
[0093] The term "sortase" refers to an enzyme that has transamidase
activity. Sortases, also referred to as transamidases, can form a
peptide linkage (i.e., amide linkage) between an appropriate acyl
donor compound and a nucleophilic acyl acceptor containing a
NH.sub.2--CH.sub.2-moiety, such as an N-terminal glycine. Sortases
recognize substrates comprising a sortase recognition motif, e.g.,
the amino acid sequence LPXTG. A molecule recognized by a sortase
(i.e., comprising a sortase recognition motif) is sometimes termed
a "sortase substrate" herein. After recognition of such a motif by
sortase the catalytic residue (e.g., cysteine) in the enzyme's
active site serves as a nucleophile to cleave a peptide bond in the
motif (e.g., the peptide bond between threonine and glycine in
LXPTG). Cleavage occurs with concomitant formation of a thioacyl
intermediate between substrate and enzyme. This intermediate is
resolved by reaction with an appropriate nucleophile, thereby
creating a new bond that links the substrate to the nucleophile.
Sortases tolerate a wide variety of moieties in proximity to the
cleavage site, thus allowing for the versatile conjugation of
diverse entities so long as the substrate contains a suitably
exposed sortase recognition motif and a suitable nucleophile is
available. The terms "sortase-mediated transacylation reaction",
"sortase-catalyzed transacylation reaction", "sortase-mediated
reaction", "sortase-catalyzed reaction", "sortase reaction" and
like terms, are used interchangeably herein to refer to such a
reaction. The terms "sortase recognition motif", "sortase
recognition sequence", and "transamidase recognition sequence"
(sometimes abbreviated as "TRS") with respect to sequences
recognized by a transamidase or sortase, are used interchangeably
herein. The term "nucleophilic acceptor sequence" refers to an
amino acid sequence capable of serving as a nucleophile in a
sortase-catalyzed reaction, e.g., a sequence comprising an
N-terminal glycine (e.g., 1, 2, 3, 4, or 5 N-terminal glycines) or
in some embodiments comprising an N-terminal alanine (e.g., 1, 2,
3, 4, or 5 N-terminal alanines).
[0094] Substrates suitable for sortase-mediated conjugation can
readily be designed. For example, polypeptides can be modified to
include a sortase recognition motif at or near their C-terminus,
thereby allowing them to serve as substrates for sortase. The
sortase recognition motif need not be positioned at the very
C-terminus of a substrate but should typically be sufficiently
accessible by the enzyme to participate in the sortase reaction. In
some embodiments a sortase recognition motif is considered to be
"near" a C-terminus if there are no more than 5, 6, 7, 8, 9, 10,
12, 15, 20, or 25 amino acids between the most N-terminal amino
acid in the sortase recognition motif (e.g., L) and the C-terminal
amino acid of the polypeptide. In some embodiments there is at
least 1, 2, 3, or 4 additional amino acids C-terminal to a G or A
in a sortase recognition motif. In some embodiments at least one
additional amino acid is G or A. For example, the sortase substrate
may comprise LPXTGG, e.g., LPETGG. In some embodiments a tag (e.g.,
a 6.times.His tag or other small peptide tag) is located C-terminal
to the sortase recognition motif, optionally separated from it by a
spacer. Upon cleavage, the tag is released. The free tag may be
detected, which may be useful to monitor the progress or extent of
the reaction. In some embodiments a sortase recognition motif is
located in a flexible loop of a polypeptide. The flexible loop may
be exposed at the surface of a properly folded polypeptide. The
loop may be cleaved by a protease so as to position the sortase
recognition motif at or near the C-terminus of a resulting cleavage
product. A polypeptide comprising a sortase recognition motif may
be modified by incorporating or attaching any of a wide variety of
moieties thereto. The resulting modified polypeptide can serve as a
sortase substrate, resulting in conjugation of the moiety to the
nucleophile. Suitable nucleophiles that can be used in a sortase
reaction typically comprise a short run (e.g., 1-10) of glycine
residues, although even an alkylamine suffices to allow the
reaction to proceed. Polypeptides can be modified to comprise a
nucleophilic acceptor sequence, e.g., a sequence comprising one or
more glycines, at their N-terminus and the resulting polypeptide
may be used as a nucleophile in a sortase-catalyzed reaction. Such
a reaction can result in installation of any of a wide variety of
entities (comprising a sortase sortase recognition motif) at the
N-terminus of the polypeptide.
[0095] A "subject" may be any vertebrate organism in various
embodiments. A subject may be individual to whom an agent, cell,
substance, or composition is administered, e.g., for experimental,
diagnostic, and/or therapeutic purposes or from whom a sample is
obtained or on whom a procedure is performed. In some embodiments a
subject is a mammal, e.g. a human, non-human primate, rodent (e.g.,
mouse, rat, rabbit), ungulate (e.g., ovine, bovine, equine, caprine
species), canine, or feline. In some embodiments a subject is an
avian. In some embodiments a subject is a non-human animal that
serves as a model for a disease or disorder that affects humans. An
animal model may be used, e.g., in preclinical studies, e.g., to
assess efficacy and/or determine a suitable dose.
[0096] The term "small molecule" is used herein to refer to
molecules, whether naturally occurring or artificially created
(e.g., via chemical synthesis) that have a relatively low molecular
weight. Typically, a small molecule is an organic compound. A small
molecule may contain multiple carbon-carbon bonds, stereocenters,
and other functional groups (e.g., amines, hydroxyl, carbonyls,
heterocyclic rings, etc.). In some embodiments, a small molecule is
monomeric. In some embodiments, a small molecule has a molecular
weight of less than about 1500 g/mol. In some embodiments, a small
molecule has a molecular weight of less than about 1000 g/mol or
less than about 500 g/mol. In certain embodiments a small molecule
is a compound that has been deemed safe and effective for use as a
diagnostic or therapeutic agent in humans or animals by an
appropriate governmental agency or regulatory body.
[0097] As used herein, a "support" may be any entity or plurality
of entities having a surface to which a substance may be attached
or on which a substance may be placed. Examples, include, e.g.,
particles, slides, filters, interior wall or bottom of a vessel
(e.g., a culture vessel such as a plate or flask, well of a
microwell plate, tube), chips, etc. A support may be composed,
e.g., of glass, metal, gels (e.g., agarose), ceramics, polymers, or
combinations thereof.
[0098] The term "tumor" as used herein encompasses abnormal growths
comprising aberrantly proliferating cells. Tumors are typically
characterized by excessive cell proliferation that is not
appropriately regulated (e.g., that does not respond normally to
physiological influences and signals that would ordinarily
constrain proliferation) and may exhibit one or more of the
following properties: dysplasia (e.g., lack of normal cell
differentiation, resulting in an increased number or proportion of
immature cells); anaplasia (e.g., greater loss of differentiation,
more loss of structural organization, cellular pleomorphism,
abnormalities such as large, hyperchromatic nuclei, high
nuclear:cytoplasmic ratio, atypical mitoses, etc.); invasion of
adjacent tissues (e.g., breaching a basement membrane); and/or
metastasis. In certain embodiments a tumor is a malignant tumor,
also referred to herein as a "cancer". Malignant tumors have a
tendency for sustained growth and an ability to spread, e.g., to
invade locally and/or metastasize regionally and/or to distant
locations, whereas benign tumors often remain localized at the site
of origin and are often self-limiting in terms of growth. The term
"tumor" includes malignant solid tumors (e.g., carcinomas,
sarcomas) and malignant growths in which there may be no detectable
solid tumor mass (e.g., certain hematologic malignancies). The term
"cancer" is generally used interchangeably with "tumor" herein
and/or to refer to a disease characterized by one or more tumors,
e.g., one or more malignant or potentially malignant tumors. Cancer
includes, but is not limited to: breast cancer; biliary tract
cancer; bladder cancer; brain cancer (e.g., glioblastomas,
medulloblastomas); cervical cancer; choriocarcinoma; colon cancer;
endometrial cancer; esophageal cancer; gastric cancer;
hematological neoplasms including acute lymphocytic leukemia and
acute myelogenous leukemia; T-cell acute lymphoblastic
leukemia/lymphoma; hairy cell leukemia; chronic lymphocytic
leukemia, chronic myelogenous leukemia, multiple myeloma; adult
T-cell leukemia/lymphoma; intraepithelial neoplasms including
Bowen's disease and Paget's disease; liver cancer; lung cancer;
lymphomas including Hodgkin's disease and lymphocytic lymphomas;
neuroblastoma; melanoma, oral cancer including squamous cell
carcinoma; ovarian cancer including ovarian cancer arising from
epithelial cells, stromal cells, germ cells and mesenchymal cells;
neuroblastoma, pancreatic cancer; prostate cancer; rectal cancer;
sarcomas including angiosarcoma, gastrointestinal stromal tumors,
leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, and
osteosarcoma; renal cancer including renal cell carcinoma and Wilms
tumor; skin cancer including basal cell carcinoma and squamous cell
cancer; testicular cancer including germinal tumors such as
seminoma, non-seminoma (teratomas, choriocarcinomas), stromal
tumors, and germ cell tumors; thyroid cancer including thyroid
adenocarcinoma and medullary carcinoma. It will be appreciated that
a variety of different tumor types can arise in certain organs,
which may differ with regard to, e.g., clinical and/or pathological
features and/or molecular markers. Tumors arising in a variety of
different organs are discussed, e.g., in DeVita, Hellman, and
Rosenberg's Cancer: Principles and Practice of Oncology (Cancer:
Principles & Practice), Lippincott Williams & Wilkins;
Ninth, North American Edition edition (May 16, 2011) or in the WHO
Classification of Tumours series, 4.sup.th ed, or 3.sup.rd ed
(Pathology and Genetics of Tumours series), by the International
Agency for Research on Cancer (IARC), WHO Press, Geneva,
Switzerland.
[0099] "Treat", "treating" and similar terms refer to providing
medical and/or surgical management of a subject. Treatment may
include, but is not limited to, administering an agent or
composition (e.g., a pharmaceutical composition) to a subject.
Treatment is typically undertaken in an effort to alter the course
of a disease (which term is used to indicate any disease, disorder,
or undesirable condition warranting therapy) in a manner beneficial
to the subject. The effect of treatment may include reversing,
alleviating, reducing severity of, delaying the onset of, curing,
inhibiting the progression of, and/or reducing the likelihood of
occurrence or recurrence of the disease or one or more symptoms or
manifestations of the disease. A therapeutic agent may be
administered to a subject who has a disease or is at increased risk
of developing a disease relative to a member of the general
population. In some embodiments a therapeutic agent may be
administered to a subject who has had a disease but no longer shows
evidence of the disease. The agent may be administered e.g., to
reduce the likelihood of recurrence of evident disease. A
therapeutic agent may be administered prophylactically, i.e.,
before development of any symptom or manifestation of a disease.
"Prophylactic treatment" refers to providing medical and/or
surgical management to a subject who has not developed a disease or
does not show evidence of a disease in order, e.g., to reduce the
likelihood that the disease will occur or to reduce the severity of
the disease should it occur. The subject may have been identified
as being at risk of developing the disease (e.g., at increased risk
relative to the general population or as having a risk factor that
increases the likelihood of developing the disease.
[0100] A "variant" of a particular polypeptide or polynucleotide
has one or more alterations (e.g., additions, substitutions, and/or
deletions) with respect to a reference polypeptide or
polynucleotide, which may be referred to as the "original
polypeptide" or "original polynucleotide", respectively. An
addition may be an insertion or may be at either terminus. A
variant may be shorter or longer than the reference polypeptide or
polynucleotide. The term "variant" encompasses "fragments". A
"fragment" is a continuous portion of a polypeptide or
polynucleotide that is shorter than the reference polypeptide or
polynucleotide. In some embodiments a variant comprises or consists
of a fragment. In some embodiments a fragment or variant is at
least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 92.5%, 95%, 96%, 97%,
98%, 99%, or more as long as the reference polypeptide or
polynucleotide. In some embodiments a fragment may lack an
N-terminal and/or C-terminal portion of a reference polypeptide.
For example, a fragment may lack up to 5%, 10%, 15%, 20%, or 25% of
the length of the polypeptide from either or both ends. A fragment
may be an N-terminal, C-terminal, or internal fragment. In some
embodiments a variant polypeptide comprises or consists of at least
one domain of a reference polypeptide. In some embodiments a
variant polynucleotide hybridizes to a reference polynucleotide
under art-recognized stringent conditions, e.g., high stringency
conditions, for sequences of the length of the reference
polypeptide. In some embodiments a variant polypeptide or
polynucleotide comprises or consists of a polypeptide or
polynucleotide that is at least 50%, 60%, 70%, 80%, 90%, 95%, 96%,
97%, 98%, 99%, or more identical in sequence to the reference
polypeptide or polynucleotide over at least 20%, 30%, 40%, 50%,
60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the
reference polypeptide or polynucleotide. In some embodiments a
variant polypeptide comprises or consists of a polypeptide that is
at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more
identical in sequence to the reference polypeptide over at least
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or
100% of the reference polypeptide, with the proviso that, for
purposes of computing percent identity, a conservative amino acid
substitution is considered identical to the amino acid it replaces.
In some embodiments a variant polypeptide comprises or consists of
a polypeptide that is at least 50%, 60%, 70%, 80%, 90%, 95%, 96%,
97%, 98%, 99%, or more identical to the reference polypeptide over
at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%,
98%, 99%, or 100% of the reference polypeptide, with the proviso
that any one or more amino acid substitutions (up to the total
number of such substitutions) may be restricted to conservative
substitutions. In some embodiments a percent identity is measured
over at least 100; 200; 300; 400; 500; 600; 700; 800; 900; 1,000;
1,200; 1,500; 2,000; 2,500; 3,000; 3,500; 4,000; 4,500; or 5,000
amino acids. In some embodiments the sequence of a variant
polypeptide comprises or consists of a sequence that has N amino
acid differences with respect to a reference sequence, wherein N is
any integer between 1 and 10 or between 1 and 20 or any integer up
to 1%, 2%, 5%, or 10% of the number of amino acids in the reference
polypeptide, where an "amino acid difference" refers to a
substitution, insertion, or deletion of an amino acid. In some
embodiments a difference is a conservative substitution.
Conservative substitutions may be made, e.g., on the basis of
similarity in side chain size, polarity, charge, solubility,
hydrophobicity, hydrophilicity and/or the amphipathic nature of the
residues involved. In some embodiments, conservative substitutions
may be made according to Table A, wherein amino acids in the same
block in the second column and in the same line in the third column
may be substituted for one another other in a conservative
substitution. Certain conservative substitutions are substituting
an amino acid in one row of the third column corresponding to a
block in the second column with an amino acid from another row of
the third column within the same block in the second column.
TABLE-US-00001 TABLE A Aliphatic Non-polar GAP ILV Polar--uncharged
CSTM NQ Polar--charged DE KR Aromatic HPWY
[0101] In some embodiments, proline (P) is considered to be in an
individual group. In some embodiments, cysteine (C) is considered
to be in an individual group. In some embodiments, proline (P) and
cysteine (C) are each considered to be in an individual group.
Within a particular group, certain substitutions may be of
particular interest in certain embodiments, e.g., replacements of
leucine by isoleucine (or vice versa), serine by threonine (or vice
versa), or alanine by glycine (or vice versa).
[0102] In some embodiments a variant is a functional variant, i.e.,
the variant at least in part retains at least one activity of the
reference polypeptide or polynucleotide. In some embodiments a
variant at least in part retains more than one or substantially all
known activities of the reference polypeptide or polynucleotide. An
activity may be, e.g., a catalytic activity, binding activity,
ability to perform or participate in a biological function or
process, etc. In some embodiments an activity is one that has (or
the lack of which has) a detectable effect on an observable
phenotype of a cell or organism. In some embodiments an activity of
a variant may be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,
90%, 95%, or more, of the activity of the reference polypeptide or
polynucleotide, up to approximately 100%, approximately 125%, or
approximately 150% of the activity of the reference polypeptide or
polynucleotide, in various embodiments. In some embodiments a
variant, e.g., a functional variant, comprises or consists of a
polypeptide at least 80%, 90%, 92.5%, 95%, 96%, 97%, 98%, 99%.
99.5% or 100% identical to an reference polypeptide or
polynucleotide over at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%, 98%, or 99% or 100% of the full length of the reference
polypeptide or polynucleotide or over at least 70%, 75%, 80%, 85%,
90%, 92.5%, 95%, 96%, 97%, 98%, or 99% or 100% of a functional
fragment of the reference polypeptide or polynucleotide. In some
embodiments an alteration, e.g., a substitution or deletion, e.g.,
in a functional variant, does not alter or delete an amino acid or
nucleotide that is known or predicted to be important for an
activity, e.g., a known or predicted catalytic residue or residue
involved in binding a substrate or cofactor. In some embodiments
nucleotide(s), amino acid(s), or region(s) exhibiting lower degrees
of conservation across species as compared with other amino acids
or regions may be selected for alteration. Variants may be tested
in one or more suitable assays to assess activity. In certain
embodiments a polypeptide or polynucleotide sequence in the NCBI
RefSeq database may be used as a reference sequence. In some
embodiments a variant or fragment of a naturally occurring
polypeptide or polynucleotide is a naturally occurring variant or
fragment. In some embodiments a variant or fragment of a naturally
occurring polypeptide or polynucleotide is not naturally occurring.
Calculations of sequence identity can be performed as follows.
Sequences are aligned for optimal comparison purposes and gaps can
be introduced in one or both of a first and a second sequence for
optimal alignment. When a position in the first sequence is
occupied by the same residue as the corresponding position in the
second sequence, the sequences are deemed to be identical at that
position. The percent identity between the two sequences is a
function of the number of identical positions shared by the
sequences, taking into account the number of gaps, and the length
of each gap, introduced for optimal alignment of the two sequences.
Sequences can be aligned and/or percent identity determined with
the use of a variety of algorithms and computer programs known in
the art. For example, computer programs such as BLAST2, BLASTN,
BLASTP, Gapped BLAST, etc., may be used to generate alignments
and/or to obtain a percent identity. The algorithm of Karlin and
Altschul (Karlin and Altschul, Proc. Natl. Acad. Sci. USA
87:22264-2268, 1990) modified as in Karlin and Altschul, Proc.
Natl. Acad Sci. USA 90:5873-5877, 1993 is incorporated into the
NBLAST and XBLAST programs of Altschul et al. (Altschul, et al., J.
MoI. Biol. 215:403-410, 1990). In some embodiments, to obtain
gapped alignments for comparison purposes, Gapped BLAST is utilized
as described in Altschul et al. (Altschul, et al. Nucleic Acids
Res. 25: 3389-3402, 1997). When utilizing BLAST and Gapped BLAST
programs, the default parameters of the respective programs may be
used. See the Web site having URL www.ncbi.nlm.nih.gov and/or
McGinnis, S. and Madden, T L, W20-W25 Nucleic Acids Research, 2004,
Vol. 32, Web server issue. Other suitable programs include CLUSTALW
(Thompson J D, Higgins D G, Gibson T J, Nuc Ac Res, 22:4673-4680,
1994) and GAP (GCG Version 9.1; which implements the Needleman
& Wunsch, 1970 algorithm (Needleman S B, Wunsch C D, J Mol
Biol, 48:443-453, 1970.) The percent identity between a sequence of
interest A and a second sequence B may be computed by aligning the
sequences, allowing the introduction of gaps to maximize identity,
determining the number of residues (nucleotides or amino acids)
that are opposite an identical residue, dividing by the minimum of
TGA and TGB (here TGA and TGB are the sum of the number of residues
and internal gap positions in sequences A and B in the alignment),
and multiplying by 100. Percent identity may be evaluated over a
window of evaluation. In some embodiments a window of evaluation
may have a length of at least 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,
98%, 99%, or more, e.g., 100%, of the length of the shortest of the
sequences being compared. In some embodiments a window of
evaluation is at least 100; 200; 300; 400; 500; 600; 700; 800; 900;
1,000; 1,200; 1,500; 2,000; 2,500; 3,000; 3,500; 4,000; 4,500; or
5,000 amino acids. In some embodiments no more than 20%, 10%, 5%,
or 1% of positions in either sequence or in both sequences over a
window of evaluation are occupied by a gap. In some embodiments no
more than 20%, 10%, 5%, or 1% of positions in either sequence or in
both sequences are occupied by a gap.
[0103] A "vector" may be any of a number of nucleic acid molecules
or viruses or portions thereof that are capable of mediating entry
of, e.g., transferring, transporting, etc., a nucleic acid of
interest between different genetic environments or into a cell. The
nucleic acid of interest may be linked to, e.g., inserted into, the
vector using, e.g., restriction and ligation. Vectors include, for
example, DNA or RNA plasmids, cosmids, naturally occurring or
modified viral genomes or portions thereof, nucleic acids that can
be packaged into viral capsids, mini-chromosomes, artificial
chromosomes, transposons (e.g., Sleeping Beauty transposon), etc.
Plasmid vectors typically include an origin of replication (e.g.,
for replication in prokaryotic cells). A plasmid may include part
or all of a viral genome (e.g., a viral promoter, enhancer,
processing or packaging signals, and/or sequences sufficient to
give rise to a nucleic acid that can be integrated into the host
cell genome and/or to give rise to infectious virus). Viruses or
portions thereof that can be used to introduce nucleic acids into
cells may be referred to as viral vectors. Viral vectors include,
e.g., adenoviruses, adeno-associated viruses, retroviruses (e.g.,
lentiviruses, gamma retroviruses), vaccinia virus and other
poxviruses, herpesviruses (e.g., herpes simplex virus), and others.
Viral vectors may or may not contain sufficient viral genetic
information for production of infectious virus when introduced into
host cells, i.e., viral vectors may be replication-competent or
replication-defective. In some embodiments, e.g., where sufficient
information for production of infectious virus is lacking, it may
be supplied by a host cell or by another vector introduced into the
cell, e.g., if production of virus is desired. In some embodiments
such information is not supplied, e.g., if production of virus is
not desired. A nucleic acid to be transferred may be incorporated
into a naturally occurring or modified viral genome or a portion
thereof or may be present within a viral capsid as a separate
nucleic acid molecule. A vector may contain one or more nucleic
acids encoding a marker suitable for identifying and/or selecting
cells that have taken up the vector. Markers include, for example,
various proteins that increase or decrease either resistance or
sensitivity to antibiotics or other agents (e.g., a protein that
confers resistance to an antibiotic such as puromycin, hygromycin
or blasticidin), enzymes whose activities are detectable by assays
known in the art (e.g., .beta.-galactosidase or alkaline
phosphatase), and proteins or RNAs that detectably affect the
phenotype of cells that express them (e.g., fluorescent proteins).
Vectors often include one or more appropriately positioned sites
for restriction enzymes, which may be used to facilitate insertion
into the vector of a nucleic acid, e.g., a nucleic acid to be
expressed. An expression vector is a vector into which a desired
nucleic acid has been inserted or may be inserted such that it is
operably linked to regulatory elements (also termed "regulatory
sequences", "expression control elements", or "expression control
sequences") and may be expressed as an RNA transcript (e.g., an
mRNA that can be translated into protein or a noncoding RNA such as
an shRNA or miRNA precursor). Expression vectors include regulatory
sequence(s), e.g., expression control sequences, sufficient to
direct transcription of an operably linked nucleic acid under at
least some conditions; other elements required or helpful for
expression may be supplied by, e.g., the host cell or by an in
vitro expression system. Such regulatory sequences typically
include a promoter and may include enhancer sequences or upstream
activator sequences. In some embodiments a vector may include
sequences that encode a 5' untranslated region and/or a 3'
untranslated region, which may comprise a cleavage and/or
polyadenylation signal. In general, regulatory elements may be
contained in a vector prior to insertion of a nucleic acid whose
expression is desired or may be contained in an inserted nucleic
acid or may be inserted into a vector following insertion of a
nucleic acid whose expression is desired. As used herein, a nucleic
acid and regulatory element(s) are said to be "operably linked"
when they are covalently linked so as to place the expression or
transcription of the nucleic acid under the influence or control of
the regulatory element(s). For example, a promoter region would be
operably linked to a nucleic acid if the promoter region were
capable of effecting transcription of that nucleic acid. One of
ordinary skill in the art will be aware that the precise nature of
the regulatory sequences useful for gene expression may vary
between species or cell types, but may in general include, as
appropriate, sequences involved with the initiation of
transcription, RNA processing, or initiation of translation. The
choice and design of an appropriate vector and regulatory
element(s) is within the ability and discretion of one of ordinary
skill in the art. For example, one of skill in the art will select
an appropriate promoter (or other expression control sequences) for
expression in a desired species (e.g., a mammalian species) or cell
type. A vector may contain a promoter capable of directing
expression in mammalian cells, such as a suitable viral promoter,
e.g., from a cytomegalovirus (CMV), retrovirus, simian virus (e.g.,
SV40), papilloma virus, herpes virus or other virus that infects
mammalian cells, or a mammalian promoter from, e.g., a gene such as
EF1alpha, ubiquitin (e.g., ubiquitin B or C), globin, actin,
phosphoglycerate kinase (PGK), etc., or a composite promoter such
as a CAG promoter (combination of the CMV early enhancer element
and chicken beta-actin promoter). In some embodiments a human
promoter may be used. In some embodiments, a promoter that
ordinarily directs transcription by a eukaryotic RNA polymerase I
(a "pol I promoter"), e.g., a promoter for transcription of
ribosomal RNA (other than 5S rRNA) may be used. In some
embodiments, a promoter that ordinarily directs transcription by a
eukaryotic RNA polymerase II (a "pol II promoter") or a functional
variant thereof is used. In some embodiments, a promoter that
ordinarily directs transcription by a eukaryotic RNA polymerase III
(a "pol III promoter"), e.g., a promoter for transcription of U6,
H1, 7SK or tRNA promoter or a functional variant thereof) or a
functional variant thereof is used. One of ordinary skill in the
art will select an appropriate promoter for directing transcription
of a sequence of interest. Examples of expression vectors that may
be used in mammalian cells include, e.g., the pcDNA vector series,
pSV2 vector series, pCMV vector series, pRSV vector series, pEF1
vector series, Gateway.RTM. vectors, etc. Examples of virus vectors
that may be used in mammalian cells include, e.g., adenoviruses,
adeno-associated viruses, poxviruses such as vaccinia viruses and
attenuated poxviruses, retroviruses (e.g., lentiviruses), Semliki
Forest virus, Sindbis virus, etc. In some embodiments, regulatable
(e.g., inducible or repressible) expression control element(s),
e.g., a regulatable promoter, is/are used so that expression can be
regulated, e.g., turned on or increased or turned off or decreased.
For example, the tetracycline-regulatable gene expression system
(Gossen & Bujard, Proc. Natl. Acad. Sci. 89:5547-5551,1992) or
variants thereof (see, e.g., Allen, N, et al. (2000) Mouse Genetics
and Transgenics: 259-263; Urlinger, S, et al. (2000). Proc. Natl.
Acad. Sci. U.S.A. 97 (14): 7963-8; Zhou, X., et al (2006). Gene
Ther. 13 (19): 1382-1390 for examples) can be employed to provide
inducible or repressible expression. Other inducible/repressible
systems may be used in various embodiments. For example, expression
control elements that can be regulated by small molecules such as
artificial or naturally occurring hormone receptor ligands (e.g.,
steroid receptor ligands such as naturally occurring or synthetic
estrogen receptor or glucocorticoid receptor ligands), tetracycline
or analogs thereof, metal-regulated systems (e.g., metallothionein
promoter) may be used in certain embodiments. In some embodiments,
tissue-specific or cell type specific regulatory element(s) may be
used, e.g., in order to direct expression in one or more selected
tissues or cell types.
[0104] In some embodiments a vector is used to insert exogenous DNA
into the genome of a cell. In general, any suitable vector may be
used. In some embodiments the vector is a viral vector, e.g., a
retroviral vector such as a lentiviral vector or gamma retroviral
vector, or an adenoviral or AAV vector. In some embodiments the
vector is a plasmid, e.g., a DNA plasmid. In some embodiments, the
plasmid comprises DNA to be inserted into the genome of a cell,
wherein the DNA is located between binding sites for a transposase
("transposase binding sites") so that integration of the DNA can be
achieved by supplying the transposase, e.g., by expressing it from
the same or a different plasmid. In some embodiments the
transposase is e.g., a member of the Sleeping Beauty family of
transposases, the piggyBac family of transposases, or the Tol2
family of transposases (see Grabundzija, I., et al., Molecular
Therapy, vol. 18 no. 6, 1200-1209 (2010) for review of transposon
systems that utilize these transposases, and various uses thereof
in genetic engineering). Examples of Sleeping Beauty transposases
include SB10, SB11, and SB100X (see, e.g., Mates, L., et al., Nat
Genet. (2009) 41(6):753-61; Jin, Z., et al. Gene Therapy (2011) 18,
849-856). In some embodiments the vector is suitable for use to
genetically engineer cells, e.g., human cells, that are to be
administered to a human subject. In some embodiments the vector has
been used in at least one clinical trial in human subjects, results
of which have been published, without reported clinically
unacceptable adverse events attributable to the vector. In some
embodiments the vector is a self-inactivating retroviral vector.
Such vectors may be created by deletion of at least part of the U3
portion of the 3' LTR. Exemplary retroviral and lentiviral vectors
are described in US Pat. Pub. No. 20050251872, US Pat. Pub. No.
20040259208, and various other references cited herein. In some
embodiments a second or third generation lentiviral vector may be
used.
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
I. Sortagging Non-Genetically Engineered Eukaryotic Polypeptides
and Cells
[0105] The present disclosure describes the unexpected discovery
that non-genetically engineered mammalian cells can be effectively
labeled using sortase, i.e., in a sortase-catalyzed transacylation.
In some aspects, the invention provides methods of using sortase to
conjugate agents to living mammalian cells that have not been
genetically engineered to express a protein comprising a sortase
recognition motif or a nucleophilic acceptor sequence. In some
embodiments the mammalian cells have not been genetically
engineered. Some aspects of this invention relate to the
recognition that the sortase-catalyzed transacylation reaction
allows for the conjugation of agents to one or more polypeptides
that are endogenous to living mammalian cells, i.e., sortase can be
used to conjugate agents to living mammalian cells that are not
genetically engineered for sortagging. As used herein, a
polypeptide is "not genetically engineered for sortagging" if the
polypeptide is not genetically engineered in a way that allows it
to serve as a sortase substrate or as a nucleophile in a
sortase-catalyzed reaction, i.e., the polypeptide is not
genetically engineered to comprise a sortase recognition motif in a
region accessible to a sortase (e.g., at or near the C-terminus)
and is not genetically engineered to comprise a nucleophilic
acceptor sequence that can serve as a nucleophile in a
sortase-catalyed reaction, such as a sequence comprising one or
more glycines, located at the N-terminus of the polypeptide or
positioned such that cleavage of the polypeptide can result in the
sequence being located at an N-terminus. In some embodiments the
polypeptide is not genetically engineered. A cell is considered
"not genetically engineered for sortagging" if the cell has not
been genetically engineered to express a polypeptide that (either
naturally or as a result of genetic engineering) is suitable to
serve as a sortase substrate or as a nucleophile in a
sortase-catalyzed reaction. In some embodiments the cell has not
been genetically engineered to express a polypeptide comprising a
sortase recognition motif or a nucleophilic acceptor sequence. In
some embodiments the cell is not genetically engineered. In some
embodiments the cell does not comprise a modification to its genome
introduced by the hand of man. In some aspects, the invention
relates to use of sortase to attach any of a wide variety of agents
to the surface of non-genetically engineered mammalian cells.
Unless otherwise indicated or clearly evident from the context,
where the present disclosure refers to sortagging mammalian cells
it is generally intended to mean mammalian cells that have not been
genetically engineered for sortagging. In certain embodiments the
animal cells are not genetically engineered.
[0106] As described in Examples 1 and 2, non-genetically engineered
mouse splenocytes were effectively sortagged with a variety of
sortase substrates at readily detectable levels. In other
experiments, sortagging of non-genetically engineered cells of a
human kidney cell line (HEK293T cells) and canine kidney cell line
(MDCK cells) and variety of other non-genetically engineered
eukaryotic cell types (fungal, protozoal) was also observed. Thus,
sortase can be used for modification of mammalian cell surfaces and
other eukaryotic cell surfaces without requiring that the cells be
engineered to express polypeptides comprising a sortase recognition
sequence or nucleophilic acceptor sequence. In accordance with
certain embodiments of the present invention a non-genetically
engineered mammalian cell expresses one or more endogenous
polypeptides comprising a nucleophilic acceptor sequence, thus
allowing it to serve as a nucleophile in a sortase-catalyzed
transacylation. Such polypeptide(s) may comprise a sequence of one
or more glycines exposed at the cell surface, e.g., in an
N-terminal domain, available to act as a nucleophile in a reaction
in which sortase is used to conjugate a sortase substrate to the
polypeptide. In some embodiments the endogenous polypeptide may
comprise an N-terminal glycine. In some embodiments the endogenous
polypeptide may comprise a sequence of between 1-10 glycines at its
N-terminus, e.g., 1, 2, 3, 4, or 5 glycines. In some embodiments
the polypeptide may not have an N-terminal glycine when initially
synthesized (e.g., the N-terminal amino acid may be methionine) but
undergoes co-translational or post-translational processing (e.g.,
cleavage) or partial degradation so that a sequence of one or more
glycines is present at the N-terminus. For example, a secretion
signal sequence may be removed. Such processing or degradation may
occur before exposure of at least a portion of the polypeptide at
the cell surface (e.g., in the endoplasmic reticulum) or may occur
following exposure of at least a portion of the polypeptide to the
extracellular environment. It will thus be understood that aspects
of the invention comprise sortagging eukaryotic cell surfaces,
e.g., mammalian cell surfaces, without first modifying the cells so
as to cause them to have a sortase recognition sequence or a moiety
capable of serving as a nucleophile in a sortase-catalyzed reaction
attached to their surface. Aspects of the invention comprise
sortagging an endogenous polypeptide expressed by a living
eukaryotic cell, e.g., a mammalian cell, wherein the endogenous
polypeptide comprises an extracellular domain that naturally
comprises one or more amino acids capable of serving as a
nucleophile in a sortase-catalyzed reaction. According to such
aspects the natural DNA sequence encoding the extracellular domain
of such polypeptide has not been modified by the hand of man to
encode an amino acid capable of serving as a nucleophile in a
sortase-catalyzed reaction, and the polypeptide has not been
modified by the hand of man by adding such an amino acid to the
extracellular domain. For example, the extracellular domain of the
polypeptide has not been subjected to covalent or noncovalent
linkage of a (G)n moiety, an (A)n moiety, or a moiety comprising a
free amine capable of serving as a nucleophile in a
sortase-catalyzed reaction. In certain embodiments of any aspect,
cells are not subjected to chemical modification prior to
sortagging.
[0107] In some aspects, the invention provides compositions useful
for generating sortase-modified eukaryotic cells, e.g.,
sortase-modified mammalian cells. In some embodiments the
compositions comprise a sortase and one or more living eukaryotic
cells, e.g., mammalian cells, wherein the cell(s) do not express a
polypeptide that has been genetically engineered to comprise a
sortase recognition motif or nucleophilic acceptor sequence. In
some embodiments the cell(s) are not genetically engineered. In
some embodiments a composition further comprises a sortase
substrate. In some embodiments the sortase substrate comprises any
of a variety of agents, e.g.,
[0108] In some aspects, the invention provides compositions
comprising sortase-modified eukaryotic cells, e.g.,
sortase-modified mammalian cells. In some embodiments the
compositions comprise one or more sortase-modified eukaryotic
cells, e.g., sortase-modified mammalian cells, wherein the cells
are modified by conjugation of an agent to a polypeptide expressed
by the cells, wherein the polypeptide has not been genetically
engineered to comprise a sortase recognition motif or nucleophilic
acceptor sequence. In some embodiments the eukaryotic cells, e.g.,
mammalian cells, are not genetically engineered.
[0109] In some aspects, the invention provides a eukaryotic cell,
e.g., a mammalian cell, that comprises an agent conjugated via a
sortase recognition motif to a non-genetically engineered
endogenous polypeptide expressed by the cell. In some embodiments
the cell is not genetically engineered. In some embodiments, two,
three, four or more different non-genetically engineered endogenous
polypeptides expressed by the cell have an agent conjugated thereto
via a sortase recognition motif. The agents attached to different
polypeptides may be the same or the cell may be sortagged with
multiple different agents.
[0110] In some aspects, the invention provides methods of
generating sortase-modified eukaryotic cells, e.g., mammalian
cells. In some aspects, the invention provides methods that
comprise conjugating an agent to a non-genetically engineered
eukaryotic, e.g., mammalian, polypeptide using a sortase. In some
aspects, the invention provides methods comprising conjugating an
agent to a mammalian polypeptide using a sortase, wherein the
polypeptide has not been engineered to comprise a sortase
recognition motif or nucleophilic acceptor sequence. In some
embodiments the polypeptide is expressed by a living mammalian
cell, and the methods comprise contacting the cell with a sortase
and a sortase substrate comprising the agent under conditions
suitable for a sortase reaction to occur. In some embodiments the
polypeptide comprises an extracellular domain, and the methods
comprise conjugating the sortase substrate to the extracellular
domain of the polypeptide. In some embodiments the extracellular
domain comprises the N-terminus of the polypeptide. In some
embodiments the mammalian polypeptide comprises an N-terminal
nucleophilic acceptor sequence, e.g., a sequence comprising an
N-terminal glycine, before conjugation of the sortase substrate
thereto. In some embodiments a method comprises contacting one or
more living mammalian cells with sortase and a sortase substrate
under conditions and for a time suitable for a sortase-mediated
transacylation reaction to occur, wherein the living mammalian
cell(s) have not been genetically engineered to express a
polypeptide that comprises a sortase recognition motif or
nucleophilic acceptor sequence. In some embodiments the mammalian
cell(s) have not been genetically engineered.
[0111] In some embodiments a method further comprises separating
one or more of the living mammalian cell(s) from sortase and/or
from sortase substrate that is not conjugated to the cells. The
cells may be processed so as to achieve a selected degree of purity
with respect to sortase, unconjugated sortase substrate, or both.
For example, in some embodiments the amount of sortase and/or the
amount of unconjugated sortase substrate may be reduced to below a
selected concentration and/or a selected proportion of the sortase
and/or unconjugated sortase substrate may be removed. For example,
the concentration of sortase and/or unconjugated sortase substrate
may be reduced by at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%,
99.5%, or more 99.9% relative to the initial concentration, or at
least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or more
of the sortase and/or unconjugated sortase substrate that was
present in the composition comprising one or more living mammalian
cells and sortase may be removed. In some embodiments the
concentration of sortase and/or unconjugated sortase substrate is
reduced to no more than 0.01%, 0.05%, 0.1%, 0.5%, or 1.0% relative
to the concentration used to sortag the mammalian cells. In some
embodiments a sortase polypeptide and/or unconjugated sortase
substrate is not detectable in the composition as measured by
standard immunoblot using an antibody or other affinity agent that
specifically binds to the polypeptide and/or agent. Various
suitable methods for separating sortagged cells from sortase and/or
from unconjugated sortase substrate are described herein, but other
suitable methods may be used.
[0112] In some aspects, the invention provides living mammalian
cells having an agent conjugated thereto via a sortase-mediated
transacylation reaction ("sortagged cells"). In some embodiments
the agent is conjugated to a polypeptide comprising a domain
exposed at the cell surface. In some embodiments compositions
comprising a plurality of such cells are provided. In some
embodiments the polypeptide to which the agent is conjugated
comprises, after such conjugation, a sortase recognition motif. In
some embodiments a composition comprising a plurality of such
sortagged cells has a reduced level of sortase, unconjugated
sortase substrate, or both, as compared with a composition in which
the cells were sortagged. In some embodiments the composition has a
selected degree of purity with respect to sortase, unconjugated
sortase substrate, or both. In some embodiments at least a selected
percentage of the cells in a composition are modified, i.e., have
an agent conjugated thereto by sortase. For example, in some
embodiments at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%,
70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more of the cells have
an agent conjugated thereto. In some embodiments a method may
comprise separating cells that have an agent conjugated thereto
from cells that do not.
[0113] In certain embodiments an endogenous mammalian polypeptide
comprises one or more N-terminal glycines. A polypeptide comprising
one or more N-terminal glycines may be represented as
G(G)n-B.sup.1, wherein G is glycine, B.sup.1 represents an amino
acid sequence, and n is a non-negative integer, e.g., between 0 and
10, or may equivalently be represented as follows:
##STR00001##
wherein B.sup.1 represents an amino acid sequence, and n is a
non-negative integer, e.g., between 0 and 10. In certain
embodiments n is 0, 1, 2, 3, 4, or 5. In general, B.sup.1 may be of
any length and sequence, provided that, in certain embodiments, the
polypeptide comprising B.sup.1 has a sequence that is endogenous to
a mammalian cell, so that a mammalian cell may express the
polypeptide without having been genetically engineered to do
so.
[0114] In some embodiments the invention provides a method
comprising contacting a living mammalian cell that comprises a
polypeptide of the following structure exposed at the cell
surface:
##STR00002##
[0115] with a sortase substrate of the following structure:
##STR00003##
[0116] wherein the transamidase recognition sequence is an amino
acid sequence motif recognized by a transamidase enzyme;
[0117] X is --O--, --NR--, or --S--; wherein R is hydrogen,
substituted or unsubstituted aliphatic, or substituted or
unsubstituted heteroaliphatic;
[0118] A.sup.1 is acyl, substituted or unsubstituted aliphatic,
substituted or unsubstituted heteroaliphatic, substituted or
unsubstituted aryl, substituted or unsubstituted heteroaryl, an
amino acid, a peptide, a protein, a polynucleotide, a carbohydrate,
a tag, a metal atom, a contrast agent, a catalyst, a
non-polypeptide polymer, a recognition element, a small molecule, a
lipid, a linker, a label, an epitope, an antigen, a therapeutic
agent, a toxin, a radioisotope, a particle;
[0119] R.sup.1 is acyl, substituted or unsubstituted aliphatic,
substituted or unsubstituted heteroaliphatic, substituted or
unsubstituted aryl, or substituted or unsubstituted heteroaryl; in
the presence of a transamidase enzyme, for example, a sortase,
under suitable conditions to form a compound of formula:
##STR00004##
wherein n is between 0 and 10, and wherein B.sup.1 represents an
extracellular domain of a polypeptide expressed by a living
mammalian cell.
[0120] The resulting sortase-modified cell may be represented as
follows:
wherein the circle represents a cell and the short line between
B.sup.1 and the cell indicates that B.sup.1 is attached to the cell
(e.g., B.sup.1 may be part of an integral membrane polypeptide or
peripheral membrane polypeptide). The sortase substrate and agent
A.sup.1 may be said to be conjugated to the cell. It will be
appreciated that the XR.sup.1 moiety of the sortase substrate is
released as a reaction byproduct.
[0121] In some embodiments X is --NR-- and XR.sup.1 represents
glycine (G), alanine (A), or another amino acid that may be found
at the C-terminus of a naturally occurring sortase recognition
sequence. In some embodiments X is --NR-- and XR.sup.1 represents
(G).sub.j(Xaa).sub.m, wherein each Xaa can be independently any
amino acid, j is at least 1, and j+m is between 1 and 5, between 1
and 10, between 1 and 20, or between 1, 5, 10, or 20 and 100. In
some embodiments j is 1. In some embodiments j is 2, 3, 4, or 5. In
some embodiments j is 1. In some embodiments j is 2, 3, 4, or 5. In
some embodiments XR.sup.1 comprises a detectable label or epitope
tag (e.g., (Xaa).sub.m may comprise an epitope tag or may have a
tag or label attached to a side chain), so that the reaction
byproduct may be detected and/or isolated or separated from the
cells.
[0122] In certain embodiments X is --O--, --NR--, or --S--; wherein
R is hydrogen, substituted or unsubstituted aliphatic, or
substituted or unsubstituted heteroaliphatic. In certain
embodiments R.sup.1 is acyl. In certain embodiments R.sup.1 is
substituted aliphatic. In certain embodiments, R.sup.1 is
unsubstituted aliphatic. In some embodiments, R.sup.1 is
substituted C.sub.1-12 aliphatic. In some embodiments, R.sup.1 is
unsubstituted C.sub.1-12 aliphatic. In some embodiments, R.sup.1 is
substituted C.sub.1-6 aliphatic. In some embodiments, R.sup.1 is
unsubstituted C.sub.1-6 aliphatic. In some embodiments, R.sup.1 is
C.sub.1-3 aliphatic. In some embodiments, R.sup.1 is butyl. In some
embodiments, R.sup.1 is n-butyl. In some embodiments, R.sup.1 is
isobutyl. In some embodiments, R.sup.1 is propyl. In some
embodiments, R.sup.1 is n-propyl. In some embodiments, R.sup.1 is
isopropyl. In some embodiments, R.sup.1 is ethyl. In some
embodiments, R.sup.1 is methyl. In certain embodiments, R.sup.1 is
substituted aryl. In certain embodiments, R.sup.1 is unsubstituted
aryl. In certain embodiments, R.sup.1 is substituted phenyl. In
certain embodiments, R.sup.1 is unsubstituted phenyl. In certain
embodiments R.sup.1 comprises a label (e.g., a fluorophore) or
affinity tag. In certain embodiments a label or affinity tag may be
used, e.g., to detect and/or remove sortase substrate that does not
participate in a sortase-mediated reaction, to detect and/or remove
reaction byproduct comprising XR.sup.1, to measure or monitor the
progress of a sortase-mediated reaction or determine the extent to
which sortase substrate has been consumed.
[0123] In certain embodiments, the C-terminal amino acid of a 5
amino acid transamidase recognition sequence, e.g., a transamidase
recognition sequence that would ordinarily comprise a C-terminal
glycine or alanine as a fifth amino acid, may be omitted. For
example, an acyl group
##STR00005##
that is not a glycine, alanine, or other residue that may be found
at the C-terminus of a naturally occurring transamidase recognition
sequence may replace the C-terminal amino acid of a 5 amino acid
transamidase recognition sequence. In some embodiments, XR.sup.1 is
selected to be a moiety that exhibits poor nucleophilicity once
released from the transamidase, thereby providing for a more
efficient ligation, e.g., as compared with the efficiency if
XR.sup.1 is a C-terminal amino acid of a naturally occurring
transamidase recognition sequence, e.g., glycine. Any moiety
exhibiting such poor nucleophilicity can be used in accordance with
certain embodiments. In some embodiments, the acyl group
##STR00006##
is not an amino acid or peptide. In some embodiments, the acyl
group is
##STR00007##
In some embodiments, the acyl group is
##STR00008##
[0124] Some embodiments of the invention provide modified,
non-genetically engineered mammalian proteins comprising a sortase
recognition motif. Some embodiments provide modified,
non-genetically engineered mammalian proteins comprising a sortase
recognition motif having an agent conjugated thereto. Some
embodiments provide mammalian cells comprising a modified,
non-genetically engineered protein comprising a sortase recognition
motif. Some embodiments provide mammalian cells comprising a
modified, non-genetically engineered protein comprising a sortase
recognition motif having an agent conjugated thereto.
[0125] Some embodiments provide a non-genetically engineered
mammalian protein comprising an N-terminal modification installed
by sortase, wherein the non-genetically engineered mammalian
protein comprises a structure according to Formula (I):
[Xaa].sub.y-TRS-PRT (I)
[0126] Some embodiments provide a non-genetically engineered
mammalian protein comprising an N-terminal modification installed
by sortase, wherein the non-genetically engineered mammalian
protein comprises a structure according to Formula (II):
M-[Xaa].sub.y-TRS-PRT (II)
In Formulas (I) and (II):
[0127] PRT is an amino acid sequence of at least three amino acids,
wherein the sequence is endogenous to a mammalian cell, e.g., a
polypeptide comprising B.sup.1 as described above;
[0128] each instance of Xaa is independently any amino acid
residue;
[0129] y is 0 or an integer between 1-2000
[0130] TRS is a transamidase recognition motif; and
[0131] M in Formula II is an agent attached to [Xaa].sub.y or, if y
is 0, M is a moiety directly attached to the TRS. In some
embodiments M comprises an amino acid, a peptide, a protein, a
polynucleotide, a carbohydrate, a tag, a metal atom, a contrast
agent, a catalyst, a non-polypeptide polymer, a recognition
element, a small molecule, a lipid, a linker, a label, an epitope,
an antigen, a therapeutic agent, a toxin, a radioisotope, a click
chemistry handle, or a particle.
[0132] In some aspects, a mammalian cell comprises a modified
protein according to Formula (I) or Formula (II). In some
embodiments at least the portion of the protein comprising
[Xaa].sub.y-TRS or comprising M-[Xaa].sub.y-TRS is exposed at the
cell surface. In some embodiments the cell is not genetically
engineered.
[0133] In some embodiments a polypeptide modified by sortase (e.g.,
a polypeptide comprising B.sup.1 as described above) is an integral
membrane protein (IMP) or a subunit of an IMP. An IMP is a protein
that is naturally stably attached to the plasma membrane of a cell.
A polypeptide may be attached to the cell in any of various ways by
which mammalian polypeptides are naturally attached to cell plasma
membranes. An IMP may comprise a transmembrane (TM) domain and, in
some embodiments an intracellular domain. A polypeptide may have
its C-terminal amino acid located within the plasma membrane or in
the cytosol. A TM polypeptide may be a single pass TM polypeptide
or multi-pass. In some embodiments a polypeptide is associated with
the membrane from one side but does not span the lipid bilayer
completely, may bind covalently to a membrane lipid, may have a
glycophosphatidylinositol (GPI) anchor, and/or may be associated
with membrane lipids via electrostatic or ionic interactions. In
some embodiments a polypeptide modified by sortase is a peripheral
membrane protein.
[0134] Sortase substrates may comprise any of a wide variety of
agents, e.g., an amino acid, a peptide, a protein, a
polynucleotide, a carbohydrate, a tag, a metal atom, a contrast
agent, a catalyst, a non-polypeptide polymer, a recognition
element, a small molecule, a lipid, a linker, a label, an epitope,
an antigen, a therapeutic agent, a toxin, a radioisotope, a
particle, or a click chemistry handle. In certain embodiments an
agent may comprise two or more such moieties. For example, a linker
may have any of a wide variety of moieties attached thereto.
[0135] In some embodiments a sortase substrate to be used to
conjugate an agent A.sup.1 to a mammalian cell using sortase may be
represented as follows:
##STR00009##
wherein X and R.sup.1 are as described above.
[0136] In some embodiments A.sup.1 comprises a protein. In some
embodiments, A.sup.1 comprises a peptide. In some embodiments,
A.sup.1 comprises an amino acid sequence comprising at least 3
amino acids. In some embodiments, A.sup.1 comprises an antibody, an
antibody chain, an antibody fragment, an antigen-binding antibody
domain, a VHH domain, a single-domain antibody, a camelid antibody,
a nanobody, an adnectin, an affibody, an anticalin, or an aptamer.
In some embodiments, A.sup.1 comprises a recombinant protein, a
protein or peptide comprising one or more non-standard amino acids
(e.g., D-amino acids), a branched protein or peptide, a therapeutic
protein or peptide, an enzyme, a polypeptide subunit of a
multisubunit protein, a transmembrane protein, a cell surface
protein, a methylated peptide or protein, an acylated peptide or
protein, a lipidated peptide or protein, a phosphorylated peptide
or protein, or a glycosylated peptide or protein. In some
embodiments, A.sup.1 comprises an antigen or an epitope. In some
embodiments A.sup.1 comprises an enzyme, growth factor, cytokine,
costimulator, or adjuvant. In some embodiments A.sup.1 comprises a
small molecule, a click chemistry handle, a fatty acid, a
polynucleotide, a carbohydrate, a tag, a metal atom, a contrast
agent, a peptide, a polypeptide, a non-polypeptide polymer, a
recognition element, a lipid, a label, or a particle. In some
embodiments A.sup.1 comprises a binding moiety. In some embodiments
A.sup.1 comprises a targeting moiety. In some embodiments a moiety
may be incorporated into A.sup.1 in any manner and at any position
that can be envisioned by those of ordinary skill in the art. For
example, A.sup.1 may comprise an amino acid, and a moiety may be
attached, e.g., to the central carbon of the amino acid, the side
chain of the amino acid, the carboxyl group of the amino acid, or
the nitrogen. In some embodiments an agent comprises an amino acid
having a side chain comprising a primary or secondary amine.
Examples of suitable amino acids include, e.g., lysine,
.epsilon.-aminocaproic acid, and various others known in the art. A
sortase recognition motif may be extended to include such an amino
acid, e.g., as K-LPXTG. Such amino acids may conveniently be used
as a point of attachment of a moiety of interest through reaction
with the amine group. In some embodiments A.sup.1 comprises a
biologically active moiety, i.e., a moiety that is capable of
causing a biological effect when contacted with a cell or
administered to a subject. In some embodiments A.sup.1 comprises or
is attached to the TRS via a linker. In some embodiments a linker
comprises a cleavage site, thereby allowing release of at least a
portion of A.sup.1 when the cleavage site is cleaved, e.g., by a
protease in vivo after administration of a sortagged cell to a
subject. In some embodiment cleavage releases an agent that
comprises both a therapeutically active or detectable moiety and a
targeting moiety. The targeting moiety may target the released
agent to a target cell or site in the body of a subject. Cleavage
may occur over a selected time frame so that agent is released over
a period of time, e.g., to maintain a therapeutically useful level
of agent over a period of time. In some embodiments the period of
time is between 12 and 24 hours, 24 and 48 hours, 2-6 days, or up
to about 1, 2, 4, 6, 10, or 12 weeks, or more.
[0137] In some aspects, the invention provides methods of using
living mammalian cells that have an agent conjugated thereto via a
sortase-catalyzed transacylation reaction (sortagged living
mammalian cells). Sortagged mammalian cells may be used in vitro,
in vivo, in research, for detection, for diagnosis, or for therapy.
Certain uses of interest are described further below but it should
be understood that the invention is not limited in this respect.
Exemplary therapeutic applications include treatment of infectious
diseases, cancer, autoimmune diseases, inflammatory conditions,
enzyme deficiencies, or immunodeficiencies. In some embodiments
sortagged living mammalian cells are used in cell therapy, e.g., in
regenerative medicine, adoptive immunotherapy, or as vaccine
components. In some embodiments sortagged mammalian cells may be
used as delivery vehicles e.g., for delivering detection agents or
therapeutic agents to a subject. In some embodiments an agent
conjugated to mammalian cells comprises a moiety that is useful in
diagnosis, monitoring, or treatment of a disease. In some
embodiments sortagged mammalian cells are administered to a
subject, e.g., a subject in need of diagnosis, monitoring, or
treatment of a disease. In some embodiments the cells originate
from a subject to whom they are subsequently administered or
originate from a donor who is histocompatible with the subject.
[0138] In some aspects, the invention provides a method of
increasing the circulation time or plasma half-life of an agent in
the body of a mammal, the method comprising: providing an agent;
and conjugating the agent to a mammalian cell using sortase. In
some embodiments the method further comprises administering the
mammalian cell to the animal, e.g., directly into the circulatory
system, e.g., intravenously. In some embodiments cells may be
administered locally, e.g., into a tissue or organ at which an
effect, e.g., a therapeutic effect, is desired. In some embodiments
conjugating an agent to a cell, e.g., a hematologic cell, e.g., a
red blood cell, lymphocyte, or red blood cell or lymphocyte
precursor, may reduce clearance of the agent, e.g., by the kidneys
and/or may reduce diffusion or transport of the agent out of the
circulatory system as compared with the rate at which the
unconjugated agent would be cleared by the kidneys or otherwise
removed from the circulatory system. In some embodiments the
mammalian cell or an ancestor thereof is obtained from a mammal to
whom the sortase-modified cell is administered. In some embodiments
the average circulation time or plasma half-life may be increased
by at least a factor of 2, 3, 5, 10, 20, 50, or more. In some
embodiments the average circulation time or plasma half-life may be
at least 5, 10, 15, 20, 25, 50 days, or more, e.g., up to the
average lifespan of the cell to which the agent is attached.
[0139] In some embodiments a therapeutic function may be provided
by a therapeutic agent, e.g., an enzyme or therapeutic antibody or
small molecule, conjugated to a mammalian cell. In some embodiments
a therapeutic function may be provided by a moiety targeting a
specific cell or cell type to a target site, attracting a specific
cell or cell type to a target site, activating a specific cell or
cell type, e.g., at a target site, stimulating or inhibiting one or
more biological activities of a specific cell or cell type, e.g.,
at a target site, providing a catalytic activity, e.g., at a target
site, or by a therapeutic agent acting on cells, e.g., at a target
site. In some embodiments a protein or other agent conjugated to
mammalian cells comprises a binding domain, e.g., an antigen
binding domain, or antibody targeting a specific cell, cell type,
tissue, or site, for example, in a subject. In some embodiments the
binding domain or antibody is conjugated to a therapeutic agent,
for example, a small molecule, or a therapeutic polypeptide. In
some embodiments the binding domain or antibody is conjugated to a
label. In some embodiments it is contemplated to attach any
therapeutic agent to mammalian cells using sortase, e.g., any
therapeutic agent known in the art.
[0140] In some embodiments a mammalian cell has an agent comprising
a label conjugated thereto, e.g., a fluorophore, fluorescent
polypeptide, quantum dot, metal-containing nanoparticle, or any
other suitable label. The mammalian cell may be detected by
detecting the label. In some embodiments the cell may be detected
in vitro, e.g., in a cell culture system. In some embodiments the
cell may be administered to a subject and detected in vivo. In some
embodiments the cell may be administered to a subject and detected
in a sample subsequently obtained from the subject. In some
embodiments the cell has a targeting moiety conjugated thereto. The
targeting moiety and label may be conjugated separately to the cell
or may be part of a single agent conjugated to the cell. The cells
may accumulate at a target site and may be detected in vivo by
detecting the label. In some embodiments the cell may further have
a therapeutic agent conjugated thereto or may provide a therapeutic
function, e.g., a cytotoxic effect against tumor cells or infected
cells.
[0141] In some embodiments a mammalian cell is sortagged with a
bifunctional agent, e.g., a bifunctional protein. In some
embodiments a bifunctional agent comprises a first domain that
provides a first function and a second domain that provides a
second function. The two domains and/or functions may be the same
or different. In some embodiments at least one domain comprises a
binding domain that targets the bifunctional agent to a target. A
target may be, e.g., an organ, a cell or cell type (e.g., a
diseased cell, such as a tumor cell or infected cell), a tissue, or
a site of disease). In some embodiments at least one domain
provides a therapeutic function or labeling function. Any of a wide
variety of bifunctional agents may be used. In some embodiments a
bifunctional agent comprises a bivalent agent, e.g., a bivalent
antibody. A bivalent agent is capable of binding to two molecules
or entities, which may be the same or different, depending on the
agent.
[0142] In some embodiments a bifunctional agent is a bispecific
agent, e.g., a bispecific protein, e.g., a bispecific antibody. In
some embodiments, a bispecific agent targets a specific antigen,
cell, cell type, or site in a cell population, tissue, organism, or
subject. For example, in some embodiments, a bispecific protein
comprises a first binding domain, e.g., an antigen binding domain,
that targets the protein to a target site (e.g., an organ, a cell
or cell type (e.g., a diseased cell, such as a tumor cell), a
tissue, or a site of disease) and a second binding domain, e.g., a
second antigen binding domain, that provides a function, e.g., a
therapeutic function. In some embodiments, a protein or binding
domain or binding agent binds to a target antigen, e.g., a tumor
antigen or an antigen of a pathogen. In some embodiments a binding
domain is conjugated to a therapeutic agent, for example, a small
molecule, or a therapeutic polypeptide. In some embodiments such
conjugation is performed using click chemistry. For example,
sortase may be used to produce a bifunctional, bivalent, or
bispecific agent by installing click chemistry handles on each of
two polypeptides (e.g., scFvs and/or sdAbs) or other molecules,
which are then conjugated to each other via a click chemistry
reaction (e.g., as discussed further below). A sortase recognition
motif may be included at or near a free C-terminus and/or a
(G).sub.n or (A).sub.n sequence may be included at or near a free
N-terminus to facilitate additional sortase-catalyzed conjugation
of the agent.
[0143] In certain embodiments an agent is a trifunctional agent. A
trifunctional agent may be a trivalent agent, e.g., a trispecific
agent. Trivalent agents or agents of even higher valency may be
produced as single polypeptides comprising three or more scFv,
sdAb, or a combination thereof. By including scFv and/or or sdAb
with different specificities in a single polypeptide chain,
multispecific (e.g., bispecific, trispecific) agents are produced.
Such agents may have multiple distinct functions conferred by
binding to different molecules or entities. Sortase may be used to
produce trifunctional agents as described for bifunctional agents.
Other methods of producing abifunctional or trifunctional agent may
also be used. For example, chemical conjugation may be performed
using any of a variety of different approaches (see, e.g.,
Hermanson, G, cited above).
[0144] In some embodiments a particle is conjugated to mammalian
cells using sortase. In some embodiments the particle comprises a
detectable label or therapeutic agent. In some embodiments the
particle is a polymeric particle. A detectable label or therapeutic
agent may be encapsulated in, impregnated into, or coated on at
least a portion of the surface of the particle or otherwise
physically associated with the particle. In some embodiments the
label or agent is released from particles over a period of time,
e.g., to maintain a therapeutically useful level of agent over a
period of time. In some embodiments the period of time is between
12 and 24 hours, 24 and 48 hours, 2-6 days, or up to about 1, 2, 4,
6, 10, or 12 weeks, or more.
[0145] In some embodiments the particle is an ultrasound
microbubble or comprises a contrast agent. In some embodiments the
particle has a diameter or average longest axis or length of at
least 5 nm, up to about 100 nm, 500 nm, 1 .mu.m, 2 .mu.m, or 3
.mu.m. In some embodiments sortagging may be used to attach a
mammalian cell to a support. The support may have an agent
comprising a TRS attached thereto, e.g., attached directly to the
support or to a coating on all or part of the support. The support
is contacted with mammalian cells and sortase under conditions such
that the agent is conjugated to the cells, thereby attaching the
cells to the support.
[0146] Those of ordinary skill in the art will understand that
sortase substrates, sortagged mammalian polypeptides, and sortagged
cells may comprise any agent, e.g., any binding agent, therapeutic
agent, or detection agent, that either comprises or can be linked
to a polypeptide comprising a sortase recognition sequence. In some
aspects, the invention encompasses mammalian cells produced
according to methods described herein, and compositions comprising
such cells, wherein the cells may be of any cell type and may have
any agent conjugated thereto using sortase. In some aspects, the
invention encompasses methods of using such cells, e.g., for one or
more purposes described herein.
[0147] In some embodiments a mammalian cell is sortagged with an
agent that is capable of binding to one or more entities. Such a
moiety may be referred to as a "binding moiety". In some
embodiments an agent comprising a binding moiety is attached to a
mammalian cell as described herein, and the mammalian cell is
placed in an environment comprising one or more entities. The
binding moiety causes the mammalian cell to become attached (bound)
to at least one of the entities via interaction between the binding
moiety and the entity. The entity may contain a specific domain,
moiety, or binding site that physically interacts with the binding
moiety. In general a binding moiety may be any moiety capable of
specifically recognizing an entity of interest. It will be
understood that a binding moiety may recognize only a portion of an
entity, e.g., an epitope of a protein. In general, a binding moiety
that binds to a particular entity may be any moiety capable of
forming appropriate interactions with the entity. In some
embodiments a binding moiety may be a protein, a peptide, an
antibody, an antibody fragment, an engineered binding protein
(e.g., an affibody, anticalin, or adnectin), a nucleic acid
aptamer, a naturally occurring or artificial ligand, etc. In some
embodiments a ligand is a small molecule. For example, a binding
moiety may be a small molecule that binds to a receptor. In some
embodiments a binding moiety may be a receptor, which may bind to
an entity comprising a ligand of the receptor. In certain
embodiments two, three, or more binding moieties with the same or
different specifities may be combined (e.g., by chemical linkage,
production as a fusion protein, or by sortase-catalyzed reactions)
to form a multivalent agent, e.g., a bivalent or trivalent agent.
In some embodiments an agent comprises a multimer or concatamer
comprising 3-10, 10-25, 25-50, 50-100, 100-1000 binding moieties,
or more. In some embodiments, a multivalent agent may have higher
affinity or avidity for a target than does an agent comprising a
single binding moiety.
[0148] In some embodiments a binding moiety is capable of targeting
an agent to a target of interest. Such a binding moiety may be
referred to as a "targeting moiety". In some embodiments an agent
comprising a targeting moiety is attached to a mammalian cell and
targets the mammalian cell to a target of interest. In general, any
binding moiety may be used as a targeting moiety, provided that the
binding moiety recognizes and binds to a target of interest. A
target of interest may be any of a variety of different entities.
In some embodiments a target of interest is associated with or
comprises a cell, structure, or molecule. In some embodiments a
target of interest is a normal cell. In some embodiments a target
of interest is an abnormal cell, e.g., a diseased cell such as a
cancer cell or infected cell. In some embodiments a targeting
moiety binds to a polypeptide, lipid, or sugar exposed at the
surface of a target cell, e.g., an extracellular domain of a
polypeptide expressed by the target cell. In some embodiments a
target of interest is or comprises a specific antigen, cell type,
or site in tissue, organ, or subject. In some embodiments a
targeting moiety binds to a marker on a cell of interest. In some
embodiments a target or marker specific for a diseased cell or site
of disease.
[0149] In some embodiments a population of mammalian cells is
contacted with two or more different sortase substrates each
comprising a sortase recognition motif and a different agent, to
produce a composition comprising cells that are sortagged with at
least two different agents. In some embodiments a population of
mammalian cells is divided into multiple aliquots. The number of
aliquots and number of cells per aliquot may be selected in any
convenient manner. In some embodiments an aliquot comprises at
least 10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6, 10.sup.7, or 10.sup.8
cells. In some embodiments the number of aliquots is between 2 and
1,000. One or more aliquots may be stored for future use. In some
embodiments two or more different sortase substrates each
comprising a sortase recognition motif and a different agent
conjugated to the sortase recognition motif, are conjugated to
cells of two or more different aliquots, to produce two or more
populations of mammalian cells having different agents conjugated
thereto. The different aliquots, or portions thereof, may be
subsequently combined. Different agents may be of the same or
different compound classes (e.g., polypeptides, polynucleotides,
small molecules). Different agents may or may not be related in
sequence or structure or capable of binding to the same target.
[0150] In some embodiments two, three, or more sequential
sortagging reactions are performed. In some embodiments, cells are
contacted with a sortase and a first substrate comprising a sortase
recognition sequence and a first agent, and the sortagging reaction
is allowed to proceed for a time and under conditions appropriate
to sortag the cells with the first agent. Cells are then separated
from the first substrate, e.g., by removing the cells or the first
substrate from the vessel. The sortagged cells are then contacted
with a second substrate comprising a sortase recognition sequence
and a second agent, and the sortagging reaction is allowed to
proceed for a time and under conditions appropriate to sortag the
cells with the second agent, resulting in cells that are sortagged
with the first agent and with a second agent. In some embodiments,
cells are contacted with a sortase and a first substrate comprising
a sortase recognition sequence and a first agent, and the
sortagging reaction is allowed to proceed for a time and under
conditions appropriate to sortag the cells with the first agent. A
second sortase substrate is then added to the reaction mixture
without separating the first sortase substrate, and the reaction is
allowed to proceed. Factors such as the time and conditions of each
sortagging reaction, the order in which different substrates are
added or used, etc., may be adjusted to achieve a desired
proportion of first and second agents attached to the cells. In
some embodiments in which two or more substrates have different
molecular weights, a substrate having a higher molecular weight may
be used before a substrate having a lower molecular weight. The
process may be repeated one or more times. If desired, a time
course may be conducted to monitor the extent of sortagging over
time. For example, conditions that result in a reaction that goes a
selected portion of the way to completion (maximum conjugation) may
be determined (e.g., 25%, 50%, 75%, 90%, or more). Information
obtained from the time course may be used to optimize the reactions
to achieve desired ratio of different agents on the cell surface.
Of course each sortagging reaction may be conducted using a mixture
of sortase substrates together, thus potentially resulting in any
number of different agents, e.g., 3, 4, 5, 6, or more, conjugated
to the cells.
[0151] In some embodiments, the total number of molecules of
agent(s) conjugated to a mammalian cell using sortase according to
the present invention is between 10 and 100; between 100 and 1,000;
between 1,000 and 10,000; between 10,000 and 50,000; between 50,000
and 100,000; between 100,000 and 500,000; between 500,000 and
1,000,000; between 1,000,000 and 2,500,000; between 2,500,000 and
5,000,000; between 5,000,000 and 10,000,000, or more. In some
embodiments, the average number of agent molecules per cell
conjugated to mammalian cells in a preparation of mammalian cells
sortagged according to the present invention is between 10 and 100;
between 100 and 1,000; between 1,000 and 10,000; between 10,000 and
50,000; between 50,000 and 100,000; between 100,000 and 500,000;
between 500,000 and 1,000,000; between 1,000,000 and 2,500,000;
between 2,500,000 and 5,000,000; between 5,000,000 and 10,000,000,
or more. The number, or average number, of molecules per cell may
be controlled, if desired, by appropriate selection of the reaction
conditions, e.g., by controlling one or more factors such as the
sortase used, the temperature, and/or the duration of the reaction.
The number, or average number, of molecules per cell may also vary
depending on the available surface area or cell type. In some
embodiments the molecules of agent(s) conjugated to a non-mammalian
cell using sortase according to the present invention is as
mentioned for mammalian cells. In certain embodiments a cell
preparation is characterized in that least 10%, 20%, 30%, 40%, 50%,
60%, 70%, 80%, 90%, or more of the cells in the cell preparation
have a number of agent molecules within any of the afore-mentioned
ranges conjugated to them.
[0152] Without wishing to be bound by any theory, sortase-mediated
modification of mammalian cells that are not genetically engineered
may have one or more advantages for a variety of purposes, e.g.,
for certain purposes in which the cells are administered to
subjects. Use of non-genetically engineered cells may, for example,
permit modification of cells that are refractory to genetic
engineering, avoid the potentially time-consuming step of genetic
engineering, and/or avoid safety concerns that may arise when
genomic sequence is modified using, e.g., viral vectors such as
retroviruses to insert a nucleic acid into the genome. Such
concerns may include potential insertional mutagenesis, which may
lead to activation of oncogenes or inactivation of tumor suppressor
genes.
[0153] While use of non-genetically engineered cells may have
certain advantages, unless otherwise indicated or clearly evident
from the context, any of the methods of generating or using
sortase-modified animal cells described herein may, in certain
embodiments, use animal cells that have been genetically engineered
to express a polypeptide that comprises a sortase recognition motif
or nucleophilic acceptor sequence, so that the polypeptide is
suitable for use as a sortase substrate or nucleophile in a
sortase-mediated reaction. Similarly, unless otherwise indicated or
clearly evident from the context, any of the compositions
comprising sortase-modified animal cells or useful for generating
or using such cells may, in certain embodiments, use cells that
have been genetically engineered to express a polypeptide that
comprises a sortase recognition motif or nucleophilic acceptor
sequence so that the polypeptide is suitable for use as a sortase
substrate or nucleophile in a sortase-mediated reaction.
[0154] In some embodiments, genetically engineered cells are
modified by using sortase to attach a sortase substrate to a
non-genetically engineered endogenous polypeptide of the cell. The
cell may, for example, have been genetically engineered to express
any of a wide variety of products, e.g., polypeptides or noncoding
RNAs, may be genetically engineered to have a deletion of at least
a portion of one or more genes, and/or may be genetically
engineered to have one or more precise alterations in the sequence
of one or more endogenous genes. In certain embodiments a
non-engineered endogenous polypeptide of such genetically
engineered cell is sortagged with any of the various agents
described herein.
[0155] Although the invention is described herein mainly in regard
to mammalian cells, the invention provides embodiments in which any
eukaryotic cell, e.g., any animal cells, e.g., any vertebrate
cells, e.g., avian cells, fish cells, amphibian cells, or reptilian
cells, or invertebrate animal cells, e.g., insect cells, or fungal
cells (e.g., yeast), or protozoal cells may be used, in any aspect
described herein. Accordingly, where the disclosure refers to
mammalian cells, it should be understood that analogous aspects and
embodiments pertaining to other eukaryotic cell types, e.g.,
fungal, insect, protozoal, are provided unless otherwise indicated
or evident from the context. In some embodiments polypeptides
endogenous to such cells may be sortagged.
[0156] In certain embodiments, sortagging eukaryotic cells, e.g.,
animal cells, as described herein does not comprise and/or is not
performed in connection with sortagging cells that have been
genetically engineered for sortagging. For example, the method is
not performed as a negative control in connection with sortagging
cells that have been genetically engineered to comprise a protein
comprising a sortase recognition sequence or nucleophilic acceptor
sequence. In certain embodiments the method is performed in order
that the sortagged animal cells that have not been engineered for
sortagging may be used for one or more purposes of interest. In
certain embodiments the sortagging occurs at a level above what
would reasonably be expected as background level of nonspecific
binding of a sortase substrate to an animal cell. In some
embodiments the number of sortagged cells produced is sufficient to
administer a therapeutically effective amount of an agent to a
mammalian subject, e.g., a human.
II. Suitable Transamidase Enzymes and Transamidase Recognition
Motifs
[0157] Enzymes identified as "sortases" have been isolated from a
variety of Gram-positive bacteria. In nature, these enzymes
catalyze a cell wall sorting reaction in which a surface protein
with a sorting signal containing a sortase recognition motif is
cleaved and the carboxyl end of the protein is covalently attached
to a pentaglycine cross-bridge of peptidoglycan. Gram-positive
bacteria include the following genera: Actinomyces, Bacillus,
Bifidobacterium, Cellulomonas, Clostridium, Corynebacterium,
Micrococcus, Mycobacterium, Nocardia, Staphylococcus,
Streptococcus, and Streptomyces. In certain embodiments the
transpeptidation reaction catalyzed by sortase results in the
ligation of species containing a sortase recognition motif with
species bearing one or more N-terminal glycine residues or an
N-terminal alkylamine group. Sortases, sortase-mediated
transacylation reactions, and their use in protein engineering are
well known to those of ordinary skill in the art (see, e.g., Ploegh
et al., International Patent Applications PCT/US2010/000274
(WO/2010/087994), and PCT/US2011/033303 (WO/2011/133704).
[0158] Additional description of use of sortase, sortase
preparation methods, sortases, sortase substrates, sortase
recognition sequences, etc., may be found in Popp M W, Ploegh H L.,
Angew Chem Int Ed Engl, 2011; 50:5024-5032; Strijbis, K., et al.,
Traffic 2012; 13: 780-789; Witte M D, et al., Proc Natl Acad Sci
USA. 2012; 109(30):11993-8; Hess G T, et al., Bioconjug Chem. 2012
Jul. 18; 23(7):1478-87, Witte M D et al. (2012) PNAS
109:11993-11998; Guimaraes C P et al. (2013) Site-specific
C-terminal and internal loop labeling of proteins using
sortase-mediated reactions. Nat Protoc 8:1787-1799, and references
in any of these.
[0159] Sortases have been classified into 4 classes, designated A,
B, C, and D, based on sequence alignment and phylogenetic analysis
of 61 sortases from Gram positive bacterial genomes (Dramsi S,
Trieu-Cuot P, Bierne H, Sorting sortases: a nomenclature proposal
for the various sortases of Gram-positive bacteria. Res Microbiol.
156(3):289-97, 2005. These classes correspond to the following
subfamilies, into which sortases have also been classified by
Comfort and Clubb (Comfort D, Clubb R T. A comparative genome
analysis identifies distinct sorting pathways in gram-positive
bacteria. Infect Immun., 72(5):2710-22, 2004): Class A (Subfamily
1), Class B (Subfamily 2), Class C (Subfamily 3), Class D
(Subfamilies 4 and 5). The aforementioned references disclose
numerous sortases and recognition motifs. See also Pallen, M. J.;
Lam, A. C.; Antonio, M.; Dunbar, K. TRENDS in Microbiology, 2001,
9(3), 97-101. Those skilled in the art will readily be able to
assign a sortase to the correct class based on its sequence and/or
other characteristics such as those described in Drami, et al.,
supra. The term "sortase A" is used herein to refer to a class A
sortase, usually named SrtA in any particular bacterial species,
e.g., SrtA from S. aureus or S. pyogenes Likewise "sortase B" is
used herein to refer to a class B sortase, usually named SrtB in
any particular bacterial species, e.g., SrtB from S. aureus. The
present disclosure encompasses embodiments relating to any of the
sortase classes known in the art (e.g., a sortase A from any
bacterial species or strain, a sortase B from any bacterial species
or strain, a class C sortase from any bacterial species or strain,
and a class D sortase from any bacterial species or strain). In
certain embodiments a sortase that utilizes a nucleophilic acceptor
sequence having an N-terminal glycine, e.g., 1-5 N-terminal
glycines, is used, such as SrtA from S. aureus. In some embodiments
it is contemplated to use two or more sortases. In some embodiments
the sortases may utilize different sortase recognition sequences
and/or different nucleophilic acceptor sequences. For example, SrtA
from S. pyogenes can utilize a nucleophilic acceptor sequence
having one or more N-terminal alanines, e.g., 1-5 N-terminal
alanines and/or may utilize a sortase recognition motif comprising
LPXTA.
[0160] Amino acid sequences of Srt A and Srt B and the nucleotide
sequences that encode them are known to those of skill in the art
and are disclosed in a number of references cited herein, the
entire contents of all of which are incorporated herein by
reference. The amino acid sequences of S. aureus SrtA and SrtB are
homologous, sharing, for example, 22% sequence identity and 37%
sequence similarity. The amino acid sequence of a
sortase-transamidase from Staphylococcus aureus also has
substantial homology with sequences of enzymes from other
Gram-positive bacteria, and such transamidases can be utilized in
the ligation processes described herein. For example, for SrtA
there is about a 31% sequence identity (and about 44% sequence
similarity) with best alignment over the entire sequenced region of
the S. pyogenes open reading frame. There is about a 28% sequence
identity with best alignment over the entire sequenced region of
the A. naeslundii open reading frame. It will be appreciated that
different bacterial strains may exhibit differences in sequence of
a particular polypeptide, and the sequences herein are
exemplary.
[0161] In certain embodiments a transamidase bearing 18% or more
sequence identity, 20% or more sequence identity, or 30% or more
sequence identity with the S. aureus, S. pyogenes, A. naeslundii,
S. nutans, E. faecalis or B. subtilis open reading frame encoding a
sortase can be screened, and enzymes having transamidase activity
comparable to Srt A or Srt B from S. aureas can be utilized (e. g.,
comparable activity sometimes is 10% of Srt A or Srt B activity or
more).
[0162] Thus in some embodiments of the invention the sortase is a
sortase A (SrtA). SrtA recognizes the motif LPXTG, with common
recognition motifs being, e.g., LPKTG, LPATG, LPNTG. In some
embodiments LPETG is used. However, motifs falling outside this
consensus may also be recognized. For example, in some embodiments
the motif comprises an `A` rather than a `T` at position 4, e.g.,
LPXAG, e.g., LPNAG. In some embodiments the motif comprises an `A`
rather than a `G` at position 5, e.g., LPXTA, e.g., LPNTA. In some
embodiments the motif comprises a `G` rather than `P` at position
2, e.g., LGXTG, e.g., LGATG. In some embodiments the motif
comprises an `I` rather than `L` at position 1, e.g., IPXTG, e.g.,
IPNTG or IPETG.
[0163] In some embodiments, a variant of a naturally occurring
sortase may be used. Such variants may be produced through
processes such as directed evolution, site-specific modification,
etc. Considerable structural information regarding sortase enzymes,
e.g., sortase A enzymes, is available, including NMR or crystal
structures of SrtA alone or bound to a sortase recognition sequence
(see, e.g., Zong Y, et al. J. Biol Chem. 2004, 279, 31383-31389).
Three dimensional structure information is also available for other
sortases, e.g., S. pyogenes SrtA (Race, P R, et al., J Biol Chem.
2009, 284(11):6924-33). The active site and substrate binding
pocket of S. aureus SrtA have been identified. One of ordinary
skill in the art can generate functional variants by, for example,
avoiding deletions or substitutions that would disrupt or
substantially alter the active site or substrate binding pocket of
a sortase. In some embodiments a functional variant of S. aureus
SrtA comprises His at position 120, Cys at position 184, and Arg at
position 197, wherein Cys at position 184 is located within a TLXTC
motif. Functional variants of other SrtA proteins may have His,
Cys, Arg, and TLXTC motifs at positions that correspond to the
positions of these residues in S. aureus SrtA. In some embodiments,
a sortase variant comprises a sequence at least 80%, 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a wild type
sortase A sequence or catalytic domain thereof, e.g., at least 80%,
85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical
to amino acids 60-206 of SEQ ID NO: 1 or SEQ ID NO: 2, or at least
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identical to amino acids 26-206 of SEQ ID NO: 1 or SEQ ID NO: 2. In
some embodiments, a sortase variant comprises 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions relative
to amino acids 60-206 of SEQ ID NO: 1 or relative to amino acids
26-206 of SEQ ID NO: 1 or SEQ ID NO: 2.
[0164] In some embodiments, a transamidase having higher
transamidase activity than a naturally occurring sortase may be
used. In some embodiments the activity of the transamidase is at
least about 10, 15, 20, 40, 60, 80, 100, 120, 140, 160, 180, or 200
times as high as that of S. aureus sortase A. In some embodiments
the activity is between about 10 and 50 times as high as that of S.
aureus sortase A, e.g., between about 10 and 20 times as high,
between about 20 and 30 times as high, between about 30 and 50
times as high. In some embodiments the activity is between about 50
and about 150 times as high as that of S. aureus sortase A, e.g.,
between about 50 and 75 times as high, between about 75 and 100
times as high, between about 100-125 times as high, or between
about 125 and 150 times as high. For example, variants of S. aureus
sortase A with up to a 140-fold increase in LPETG-coupling activity
compared with the starting wild-type enzyme have been identified
(Chen, I., et al., PNAS 108(28): 11399-11404, 2011). In some
embodiments such a sortase variant is used in a composition or
method of the invention. In some embodiments a sortase variant
comprises any one or more of the following substitutions relative
to a wild type S. aureus SrtA: P94S or P94R, D160N, D165A, K190E,
and K196T mutations.
[0165] One of ordinary skill in the art will appreciate that the
foregoing descriptions of substitutions utilize standard notation
of the form X.sub.1NX.sub.2, in which X.sub.1 and X.sub.2,
represent amino acids and N represents an amino acid position,
X.sub.1 represents an amino acid present in a first sequence (e.g.,
a wild type S. aureus SrtA sequence), and X.sub.2 represents an
amino acid that is substituted for X.sub.1 at position N, resulting
in a second sequence that has X.sub.2 at position N instead of
X.sub.1. It should be understood that the present disclosure is not
intended to be limited in any way by the identity of the original
amino acid residue X.sub.1 that is present at a particular position
N in a wild type SrtA sequence used to generate a SrtA variant and
is replaced by X.sub.2 in the variant. Any substitution which
results in the specified amino acid residue at a position specified
herein is contemplated by the disclosure. Thus a substitution may
be defined by the position and the identity of X.sub.2, whereas
X.sub.1 may vary depending, e.g., on the particular bacterial
species or strain from which a particular SrtA originates. Thus in
some embodiments, a sortase A variant comprises any one or more of
the following: an S residue at position 94 (S94) or an R residue at
position 94 (R94), an N residue at position 160 (N160), an A
residue at position 165 (A165), an E residue at position 190
(E190), a T residue at position 196 (T196) (numbered according to
the numbering of a wild type SrtA, e.g., SEQ ID NO: 1). For
example, in some embodiments a sortase A variant comprises two,
three, four, or five of the afore-mentioned mutations relative to a
wild type S. aureus SrtA (e.g., SEQ ID NO: 1). In some embodiments
a sortase A variant comprises an S residue at position 94 (S94) or
an R residue at position 94 (R94), and also an N residue at
position 160 (N160), an A residue at position 165 (A165), and a T
residue at position 196 (T196). For example, in some embodiments a
sortase A variant comprises P94S or P94R, and also D160N, D165A,
and K196T. In some embodiments a sortase A variant comprises an S
residue at position 94 (S94) or an R residue at position 94 (R94)
and also an N residue at position 160 (N160), A residue at position
165 (A165), a E residue at position 190, and a T residue at
position 196. For example, in some embodiments a sortase A variant
comprises P94S or P94R, and also D160N, D165A, K190E, and K196T. In
some embodiments a sortase A variant comprises an R residue at
position 94 (R94), an N residue at position 160 (N160), a A residue
at position 165 (A165), E residue at position 190, and a T residue
at position 196. In some embodiments a sortase comprises P94R,
D160N, D165A, K190E, and K196T.
[0166] It is to be further understood that the disclosure
contemplates variants of any wild-type sortase A. Those skilled in
the art will appreciate that wild-type sequences of sortase A may
vary, e.g., SrtA from various species may have gaps, insertions,
and/or may vary in length relative to the amino acid sequence of
exemplary wild-type S. aureus SrtA. Those skilled in the art will
appreciate that the positions described herein in regard to
substitutions or other alterations pertain to the sequence of
exemplary wild type S. aureus SrtA, unless otherwise indicated, and
that such positions may be adjusted when making corresponding
substitutions in different bacterial SrtA sequences in order to
account for such gaps, insertions, and/or length differences. For
example, as noted above, certain sortase variants comprise a
substitution at amino acid position 94 (e.g., the amino acid is
changed to an S residue). However, the amino acid at position 94 in
S. aureus SrtA may correspond to an amino acid at a different
position (e.g., position Z) in SrtA from a second bacterial species
when the sequences are aligned. When generating a variant of the
SrtA of the second bacterial species comprising a substitution at
"position 94" (based on the wild type S. aureus SrtA sequence
numbering), it is the amino acid at position Z of the SrtA from the
second bacterial species that should be changed (e.g., to S) rather
than the amino acid at position 94. Those skilled in the art will
understand how to align any original wild-type sortase A sequence
to be used for generating a SrtA variant with an exemplary
wild-type S. aureus sortase A sequence for purposes of determining
the positions in the original wild-type sortase A sequence that
correspond to the exemplary wild-type S. aureus sortase A sequence
when taking into account gaps and/or insertions in the alignment of
the two sequences.
[0167] In some embodiments, amino acids at position 94, 160, 165,
190, and/or 196 are altered in a variant as compared with the amino
acids present at those positions in a wild type S. aureus SrtA, and
the other amino acids of the variant are identical to those present
at the corresponding positions in a wild type SrtA, e.g., a wild
type S. aureus SrtA. In some embodiments, one or more of the other
amino acids of a variant, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of
the other amino acids differ from those present at corresponding
position(s) in a wild type SrtA, e.g., a wild type S. aureus SrtA.
In some embodiments a variant may have any of the properties or
degrees of sequence identity specified in the definition of
"variants" above.
[0168] An exemplary wild type S. aureus SrtA sequence (Gene ID:
1125243, NCBI RefSeq Acc. No. NP_375640.1) is shown below, with the
afore-mentioned positions underlined:
TABLE-US-00002 (SEQ ID NO: 1)
MKKWTNRLMTIAGVVLILVAAYLFAKPHIDNYLHDKDKDEKIEQYDKNVK
EQASKDNKQQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATPEQLNRG
VSFAEENESLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNET
RKYKMTSIRDVKPTDVEVLDEQKGKDKQLTLITCDDYNEKTGVWEKRKIF VATEVK.
One of ordinary skill in the art will appreciate that different
subspecies, strains, and isolates may differ in sequence at
positions that do not significantly affect activity. For example,
another exemplary wild type S. aureus SrtA sequence (Gene ID:
3238307, NCBI RefSeq Acc. No. YP_187332.1; GenBank Acc. No.
AAD48437) has a K residue at position 57 and a G residue at
position 167, as shown below in SEQ ID NO: 2:
TABLE-US-00003 (SEQ ID NO: 2)
MKKWTNRLMTIAGVVLILVAAYLFAKPHIDNYLHDKDKDEKIEQYDKNVK
EQASKDKKQQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATPEQLNRG
VSFAEENESLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNET
RKYKMTSIRDVKPTDVGVLDEQKGKDKQLTLITCDDYNEKTGVWEKRKIF VATEVK
[0169] Either or both of these amino acids (i.e., K57 and/or G167)
may be present in or introduced into any SrtA sequence, e.g., any
S. aureus SrtA sequence, whether naturally occurring or generated
by man. Furthermore, as described herein, any sortase sequence may
further comprise a tag (e.g., 6.times.His), a spacer, or both. For
example, the N- or C-terminus may be extended to encompass a tag,
optionally separated from the rest of the sequence by a spacer,
[0170] In some embodiments a sortase variant comprising the
following sequence may be used, in which amino acid substitutions
relative to a wild type S. aureus SrtA of SEQ ID NO: 1 or SEQ ID
NO: 2 are shown in underlined bold letters:
TABLE-US-00004 (SEQ ID NO: 3)
MQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATREQLNRGVSFAEENE
SLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSI
RNVKPTAVEVLDEQKGKDKQLTLITCDDYNEETGVWETRKIFVATEVK.
[0171] As will be appreciated, amino acids 2-148 of the above
sequence correspond to amino acids 60-206 of the full length S.
aureus SrtA sequence (the catalytic domain). For example, the "R"
residue at position 36 of SEQ ID NO: 3 corresponds to the "P"
residue at position 94 in SEQ ID NO: 1 or 2. It is also
contemplated in some embodiments to use sortase variants that have
other substitutions at one or more of positions 94, 160, 165, 190,
and 196 (numbered according to the numbering of SEQ ID NO: 1 or 2),
e.g., wherein such substitutions utilize an amino acid that would
be a conservative substitution at the relevant position as compared
with the sequence of SEQ ID NO: 3.
[0172] In some embodiments a calcium-independent sortase, e.g., a
calcium-independent sortase A, is used. In some embodiments a
calcium-independent variant of S. aureus SrtA is used. As used
herein "calcium-independent" refers to the ability of a sortase
enzyme, e.g., a sortase A enzyme, to exhibit catalytic activity in
a manner that is substantially independent of the absence,
presence, or concentration of calcium, at least across a
concentration range of about 0 mM-about 10 mM. For example, in some
embodiments the activity of a calcium-independent sortase in an
aqueous medium that comprises a calcium chelator such as EDTA or
EGTA at a concentration sufficient to chelate substantially all
calcium ions is equal to or at least approximately 80%, 85%, 90%,
95%, or more as great as its activity in the same medium in the
absence of the calcium chelator. A calcium-independent sortase may
exhibit calcium-independent activity at higher calcium
concentrations as well, e.g., up to any concentration that is not
detrimental to proper functioning of the enzyme. In some
embodiments, a sortase can be assayed for ability to exhibit
sortase catalytic activity in the presence of calcium
concentrations that are lower than calcium concentrations which are
required for calcium-dependent sortase to exhibit catalytic
activity. As used herein, "calcium-dependent" in connection with a
sortase means that the catalytic activity of the sortase relies or
depends on the presence and concentration of calcium, such that in
the absence of calcium or the absence of a sufficient amount of
calcium, the calcium-dependent sortase will not exhibit sortase
catalytic activity or has greatly reduced catalytic activity (e.g.,
less than about 5%, or less than about 10%, of the activity that it
has when calcium is present in sufficient amounts (e.g., 5 mM-10
mM)). In some embodiments a calcium-dependent sortase, e.g., a wild
type S. aureus SrtA or catalytic domain thereof, is used to sortag
eukaryotic cells in a medium containing more than 0.5 mM calcium,
e.g., at least 1.0 mM, at least 2.0 mM, at least 3.0 mM, at least
4.0 mM, or at least 5.0 mM calcium. For example, the concentration
of calcium may be 1.0 mm-2.5 mM, 2.5 mM-5.0 mM, 5.0 mM-7.5 mM, 7.5
mM-10.0 mM, 10.0 mM-15 mM, 15 mM-20 mM. In some embodiments, a
calcium-independent sortase may be used to sortag eukaryotic cells
in a medium that lacks calcium or has a low calcium concentration
(e.g., a calcium concentration below that at which the sortase
exhibits maximum activity).
[0173] A sortase (e.g., a sortase having a naturally occurring
sortase sequence or a sortase variant generated by man) can be
assayed for ability to exhibit sortase catalytic activity in a
calcium-independent manner by, for example, contacting a target
protein comprising a C-terminal sortase recognition motif with a
tagged N-terminal oligoglycine derivative in the absence of calcium
in the presence of the sortase and determining whether the target
protein is ligated to the tagged N-terminal oligoglycine derivative
by the sortase. In some embodiments, catalytic activity may be
measured by the yield of sortagged target protein after a selected
time period, e.g., about 6, 12, or 18 hours of reaction. In some
embodiments, catalytic activity may be measured by measuring
k.sub.cat, K.sub.m, and/or k.sub.cat/K.sub.m. In some embodiments,
one or more kinetic parameters of SrtA activity (e.g., k.sub.cat,
k.sub.cat/K.sub.m) may be determined as described in Ton-That et
al., J Biol Chem. 2000; 275(13):9876-81. In some embodiments a
calcium-independent sortase is a variant of a calcium-dependent
sortase, wherein the variant comprises one or more amino acid
substitutions relative to the calcium-dependent sortase. In some
embodiments a calcium-independent variant has a k.sub.cat at least
about 25%, 30%, 40%, 45%, 50%, 55%, 60%, or more as high as that of
a calcium-dependent sortase of which it is a variant. In some
embodiments a calcium-independent variant has a k.sub.cat/K.sub.m
at least about 25%, 30%, 40%, 45%, 50%, 55%, 60%, or more as high
as that of a calcium-dependent sortase of which it is a
variant.
[0174] In some embodiments a calcium-independent sortase is a
naturally occurring sortase, e.g., SrtA from S. pyogenes B.
anthracis, E. faecalis, L. plantarum, L. lactis, or L.
monocytogenes. In some embodiments a calcium-independent sortase is
a S. aureus SrtA variant. In some embodiments, the present
invention contemplates use of any sortase A, e.g., any mutant of an
S. aureus SrtA, described in co-pending US provisional patent
application entitled "Calcium-Independent Sortase A Mutants",
Attorney Docket Number: WIBR-141-001, filed on even date herewith,
which is hereby incorporated by reference. It should be noted that
the term "mutant" is used interchangeably with "variant" and should
not be considered to imply that any particular way of generating
the mutant sequences is required or that any particular starting
materials is required. The disclosure contemplates any suitable
method of generating variants. Examples of suitable methods
include, but are not limited to, introducing mutations into an
appropriate wild-type coding sequence (e.g., using site-specific
mutagenesis), synthesizing the sequences of the variants de novo,
for example, utilizing solid phase peptide synthesis, and in vitro
translation a synthetic mRNA, to name only a few.
Calcium-independent sortases may be used in various embodiments of
any method or composition described herein. In some embodiments, a
calcium-independent S. aureus SrtA variant has a mutation at
position 105 and position 108 as compared with a wild type S.
aureus SrtA. For example, glutamine (E) residues at position 105
and position 108 in a wild type S. aureus SrtA sequence may be
changed to a residue that is present at the corresponding position
in a calcium-independent sortase (e.g., K or Q, respectively). In
some embodiments, for example, a sortase variant comprises an E105K
and an E108A substitution or an E105K and an E108Q substitution in
a wild type S. aureus SrtA sequence or functional variant or
fragment thereof.
[0175] In some embodiments, a calcium-independent sortase A variant
comprises at least three amino acid substitutions relative to a
wild-type sortase A, wherein the amino acid substitutions comprise
a) a K residue at position 105; b) a Q or A residue at position
108; and c) at least one amino acid substitution selected from the
group consisting of i) a R residue at position 94; ii) a S residue
at position 94; iii) a N residue at position 160; iv) a A residue
at position 165; v) a E residue at position 190; and vi) a T
residue at position 196. In some embodiments a calcium-independent
sortase A variant comprises the following amino acid substitutions
relative to a wild-type sortase A: a) a K residue at position 105;
b) a Q or A residue at position 108; c) an S residue at position 94
or R residue at position 94; d) an N residue at position 160; e) an
A residue at position 165, and a T residue at position 196. In some
embodiments, a calcium-independent sortase A variant comprises the
following amino acid substitutions relative to a wild-type sortase
A: a) a K residue at position 105; b) a Q or A residue at position
108; c) a R or S residue at position 94; d) a N residue at position
160; e) a A residue at position 165; f) a E residue at position
190; and g) a T residue at position 196. In some embodiments a
sortase comprises the following sequence, in which amino acids at
positions 94, 105, 108, 160, 165, 190, and 196 relative to a full
length S. aureus SrtA sequence are shown in bold:
TABLE-US-00005 (SEQ ID NO: 4)
MQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATREQLNRGVSFAKENQ
SLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSI
RNVKPTAVEVLDEQKGKDKQLTLITCDDYNEETGVWETRKIFVATEVK.
[0176] In some embodiments a transamidase that has an altered
substrate selectivity as compared with a naturally occurring
sortase may be used. For example, variants of S. aureus sortase A
that accept aromatic amino acids (e.g., phenylalanine), as well as
amino acids with small side chains such as Ala, Asp, Ser, Pro, and
Gly, at position 1 of the sortase recognition motif (instead of L)
have been identified (Piotukh K, et al., J Am Chem Soc.,
133(44):17536-9, 2011). In some embodiments such a sortase is used
in a composition or method of the invention. A sortase with an
altered substrate selectivity with regard to the sortase
recognition motif may be generated by engineering one or more
mutations in the sortase, e.g., in a region of the protein that is
involved in recognition and/or binding of the sortase recognition
motif, e.g., the putative substrate recognition loop (e.g., the
loop connecting strands .beta.6 and .beta.7 (.beta.6/.beta.7 loop)
in SrtA (Val161-Asp176). A crystal structure of S. aureus SrtA and
a substrate, illustrating the loops, is described in Zong, Y., et
al., J Biol Chem. 2004 Jul. 23; 279(30):31383-9.). In some
embodiments, a phage-display, yeast display, or other screen of a
mutant sortase library randomized in the substrate recognition loop
may be performed, and variants with altered substrate specificity
may be identified.
[0177] In some embodiments the sortase is a sortase B (SrtB), e.g.,
a sortase B of S. aureus, B. anthracis, or L. monocytogenes. Motifs
recognized by sortases of the B class (SrtB) often fall within the
consensus sequences NPXTX, e.g., NP[Q/K]-[T/s]-[N/G/s], such as
NPQTN or NPKTG. For example, sortase B of S. aureus or B. anthracis
cleaves the NPQTN or NPKTG motif of IsdC in the respective bacteria
(see, e.g., Marraffini, L. and Schneewind, O., Journal of
Bacteriology, 189(17), p. 6425-6436, 2007). Other recognition
motifs found in putative substrates of class B sortases are NSKTA,
NPQTG, NAKTN, and NPQSS. For example, SrtB from L. monocytogenes
recognizes certain motifs lacking P at position 2 and/or lacking Q
or K at position 3, such as NAKTN and NPQSS (Mariscotti J F, et
al., The Listeria monocytogenes sortase-B recognizes varied amino
acids at position two of the sorting motif. J Biol Chem. 2009 Jan.
7. [Epub ahead of print]).
[0178] In some embodiments, the sortase is a class C sortase. Class
C sortases may utilize LPXTG as a recognition motif.
[0179] In some embodiments, the sortase is a class D sortase.
Sortases in this class are predicted to recognize motifs with a
consensus sequence NA-[E/A/S/H]-TG (Comfort D, supra). Class D
sortases have been found, e.g., in Streptomyces spp.,
Corynebacterium spp., Tropheryma whipplei, Thermobifida fusca, and
Bifidobacterium longhum. LPXTA or LAXTG may serve as a recognition
sequence for class D sortases, e.g., of subfamilies 4 and 5,
respectively subfamily-4 and subfamily-5 enzymes process the motifs
LPXTA and LAXTG, respectively). For example, B. anthracis Sortase
C, which is a class D sortase, has been shown to specifically
cleave the LPNTA motif in B. anthracis BasI and BasH (Marrafini,
supra).
[0180] See Barnett and Scott for description of a sortase from that
recognizes QVPTGV motif (Barnett, T C and Scott, J R, Differential
Recognition of Surface Proteins in Streptococcus pyogenes by Two
Sortase Gene Homologs. Journal of Bacteriology, Vol. 184, No. 8, p.
2181-2191, 2002).
[0181] The invention contemplates use of sortases found in any gram
positive organism, such as those mentioned herein and/or in the
references (including databases) cited herein. The invention also
contemplates use of sortases found in gram negative bacteria, e.g.,
Colwellia psychrerythraea, Microbulbifer degradans, Bradyrhizobium
japonicum, Shewanella oneidensis, and Shewanella putrefaciens. They
recognize sequence motifs LP[Q/K]T[A/S]T. In keeping with the
variation tolerated at position 3 in sortases from gram positive
organisms, a sequence motif LPXT[A/S], e.g., LPXTA or LPSTS may be
used. Use of sortases from Archaea (e.g. Methanobacterium
thermoautotrophicum) is contemplated in certain embodiments.
[0182] In some embodiments, the sortase, or transamidase,
recognition sequence is LPXTG, wherein X is a standard or
non-standard amino acid. In some embodiments, X is selected from D,
E, A, N, Q, K, or R. In some embodiments, the recognition sequence
is selected from LPXTG, SPXTG, LAXTG, LSXTG, NPXTG, VPXTG, IPXTG,
and YPXRG, wherein X may be selected from D, E, A, N, Q, K, or R in
certain embodiments. In some embodiments a C-terminal G is replaced
by A. In some embodiments X is selected to match a naturally
occurring transamidase recognition sequence.
[0183] In certain embodiments the C-terminal amino acid residue of
a sortase recognition motif may be replaced with a moiety that
exhibits poorer nucleophilicity once released from the sortase
(PCT/US2010/000274; Antos, J., et al., J. Am. Chem. Soc., 2009, 131
(31), pp 10800-10801). For example, the G in LPXTG may be replaced
by a moiety that exhibits poorer nucleophilicity than glycine once
released from the sortase, such as an alkyl ester, e.g., a methyl
ester. In some embodiments, the transamidase recognition sequence
is selected from: LPKT, LPIT, LPDT, SPKT, LAET, LAAT, LAET, LAST,
LAET, LPLT, LSRT, LPET, VPDT, IPQT, YPRR, LPMT, LPLT, LAFT, LPQT,
NSKT, NPQT, NAKT, and NPQS. In certain embodiments any of the
afore-mentioned TRSs having four amino acids may further comprise a
moiety that exhibits a pooer nucleophilicity than glycine once
released from the sortase.
[0184] In some embodiments, e.g., in certain embodiments in which
sortase A is used, the transamidase recognition motif comprises the
amino acid sequence X.sub.1PX.sub.2X.sub.3 or
X.sub.1PX.sub.2X.sub.3G, where X.sub.1 is leucine, isoleucine,
valine or methionine; X.sub.2 is any amino acid; X.sub.3 is
threonine, serine or alanine; P is proline and G is glycine. In
specific embodiments, as noted above X.sub.1, is leucine and
X.sub.3 is threonine. In certain embodiments, X.sub.2 is aspartate,
glutamate, alanine, glutamine, lysine or methionine. In certain
embodiments, e.g., where sortase B is utilized, the recognition
sequence often comprises the amino acid sequence NPX.sub.1TX.sub.2,
where X.sub.1 is glutamine or lysine; X.sub.2 is asparagine or
glycine; N is asparagine; P is proline and T is threonine. In some
embodiments selection of X may be based at least in part in order
to confer desired properties on the compound containing the
recognition motif. In some embodiments, X is selected to modify a
property of the compound that contains the recognition motif, such
as to increase or decrease solubility in a particular solvent. In
some embodiments, X is selected to be compatible with reaction
conditions to be used in synthesizing a compound comprising the
recognition motif, e.g., to be unreactive towards reactants used in
the synthesis.
[0185] The invention contemplates use of sortase recognition motifs
from any of the experimentally verified or putative sortase
substrates listed at
http://bamics3.cmbi.kun.nl/jos/sortase_substrates/help.html, the
contents of which are incorporated herein by reference, and/or in
any of the above-mentioned references. In some embodiments the
sortase recognition motif is selected from: LPKTG, LPITG, LPDTA,
SPKTG, LAETG, LAATG, LAHTG, LASTG, LAETG, LPLTG, LSRTG, LPETG,
VPDTG, IPQTG, YPRRG, LPMTG, LPLTG, LAFTG, LPQTS, it being
understood that in various embodiments of the invention the
5.sup.th residue is replaced, as described elsewhere herein. For
example, the sequence used may be LPXT, LAXT, LPXA, LGXT, IPXT,
NPXT, NPQS, LPST, NSKT, NPQT, NAKT, LPIT, LAET, or NPQS. The
invention comprises embodiments in which `X` in any sortase
recognition motif disclosed herein or known in the art is any
standard or non-standard amino acid. Each variation is disclosed.
In some embodiments, X is selected from the 20 standard amino acids
found most commonly in proteins found in living organisms. In some
embodiments, e.g., where the recognition motif is LPXTG or LPXT, X
is D, E, A, N, Q, K, or R. In some embodiments, X in a particular
recognition motif is selected from those amino acids that occur
naturally at position 3 in a naturally occurring sortase substrate.
For example, in some embodiments X is selected from K, E, N, Q, A
in an LPXTG or LPXT motif where the sortase is a sortase A. In some
embodiments X is selected from K, S, E, L, A, N in an LPXTG or LPXT
motif and a class C sortase is used. In some embodiments the first
position of a sortase recognition motif is an aromatic amino acid
(e.g., F or W) or an amino acid with a relatively small side chain
such as A, D, S, P, and G, and a sortase variant capable of
recognizing the resulting motif is used. For example, in some
embodiments L in LPXT is replaced by A, D, S, P, G, F, or W.
[0186] In some embodiments, a recognition sequence further
comprises one or more additional amino acids, e.g., at the N or C
terminus. For example, one or more amino acids (e.g., up to 5 amino
acids) having the identity of amino acids found immediately
N-terminal to, or C-terminal to, a 5 amino acid recognition
sequence in a naturally occurring sortase substrate may be
incorporated. Such additional amino acids may provide context that
improves the recognition of the recognition motif.
[0187] In some embodiments any of the sortase recognition sequences
may further comprise one or more additional glycines or alanines at
the C-terminus. It will be appreciated that a C-terminal amino acid
of a polypeptide, e.g., a C-terminal amino acid of a polypeptide
comprising a transamidase recognition sequence, may be amidated,
i.e., a C-terminal amino acid may have a --CONH.sub.2 group instead
of a --COOH group at the C-terminus in certain embodiments.
[0188] The term "transamidase recognition sequence" may refer to a
masked or unmasked transamidase recognition sequence. An unmasked
transamidase recognition sequence can be recognized by a
transamidase. An unmasked transamidase recognition sequence may
have been previously masked, e.g., as described in WO2010087994. In
some embodiments, a "masked transamidase recognition sequence" is a
sequence that is not recognized by a transamidase but that can be
readily modified ("unmasked") such that the resulting sequence is
recognized by a transamidase. For example, in some embodiments at
least one amino acid of a masked transamidase recognition sequence
has a side chain that comprises a moiety that inhibits, e.g.,
substantially prevents, recognition of the sequence by a
transamidase of interest, wherein removal of the moiety allows the
transamidase to recognize the sequence. Masking may, for example,
reduce recognition by at least 80%, 90%, 95%, or more (e.g., to
undetectable levels) in certain embodiments. By way of example, in
certain embodiments a threonine residue in a transamidase
recognition sequence such as LPXTG is phosphorylated, thereby
rendering it refractory to recognition and cleavage by SrtA. The
masked recognition sequence can be unmasked by treatment with a
phosphatase, thus allowing it to be used in a SrtA-catalyzed
transamidation reaction.
[0189] It will be appreciated that transamidase fragments having
transamidation activity can be utilized in the methods described
herein. As described in PCT/US2010/000274, such fragments can be
identified by producing transamidase fragments by known recombinant
techniques or proteolytic techniques, for example, and determining
the rate of protein or peptide ligation. The fragment sometimes
consists of about 80% of the full-length transamidase amino acid
sequence, and sometimes about 70%, about 60%, about 50%, about 40%
or about 30% of the full-length transamidase amino acid sequence
such as that of S. aureus Sortase A (GenBank Accession number
AAD48437). In some embodiments, the fragment lacks an N-terminal
portion of the full-length sequence, e.g., the fragment lacks the
N-terminal portion extending to the end of the membrane anchor
sequence (up to about amino acid 26). In some embodiments the
fragment comprises the C-terminus of a full-length transamidase
amino acid sequence. In some embodiments, a catalytic core region
from a sortase is utilized, e.g., a region is from about position
60 to about position 206 of SrtA, e.g., S. aureus SrtA, or about
from position 82 to about position 249 of SrtAstrep. Thus a sortase
may comprise or consist of a catalytic domain of a full length
sortase polypeptide. It will be appreciated that the polypeptide
may also comprise an N-terminal methionine residue.
[0190] Transamidases from other organisms also can be utilized in
the processes described herein. Such transamidases often are
encoded by nucleotide sequences substantially identical or similar
to the nucleotide sequences that encode Srt A and Srt B. A similar
or substantially identical nucleotide sequence may include
modifications to the native sequence, such as substitutions,
deletions, or insertions of one or more nucleotides. Included are
nucleotide sequences that sometimes are 55%, 60%, 65%, 70%, 75%,
80%, or 85% or more identical to a native nucleotide sequence, and
often are 90% or 95% or more identical to the native nucleotide
sequence (each identity percentage can include a 1%, 2%, 3% or 4%
variance). One test for determining whether two nucleic acids are
substantially identical is to determine the percentage of identical
nucleotide sequences shared between the nucleic acids.
[0191] Calculations of sequence identity can be performed as
follows. Sequences are aligned for optimal comparison purposes and
gaps can be introduced in one or both of a first and a second
nucleic acid sequence for optimal alignment. Also, non-homologous
sequences can be disregarded for comparison purposes. The length of
a reference sequence aligned for comparison purposes sometimes is
30% or more, 40% or more, 50% or more, often 60% or more, and more
often 70%, 80%, 90%, 100% of the length of the reference sequence.
The nucleotides at corresponding nucleotide positions then are
compared among the two sequences. When a position in the first
sequence is occupied by the same nucleotide as the corresponding
position in the second sequence, the nucleotides are deemed to be
identical at that position. The percent identity between the two
sequences is a function of the number of identical positions shared
by the sequences, taking into account the number of gaps, and the
length of each gap, introduced for optimal alignment of the two
sequences.
[0192] Comparison of sequences and determination of percent
identity between two sequences can be accomplished using a
mathematical algorithm. Percent identity between two nucleotide
sequences can be determined using the algorithm of Meyers &
Miller, CABIOS 4: 11 17 (1989), which has been incorporated into
the ALIGN program (version 2.0), using a PAM120 weight residue
table, a gap length penalty of 12 and a gap penalty of 4. Percent
identity between two nucleotide sequences can be determined using
the GAP program in the GCG software package (available at
www.gcg.com), using a NWSgapdna. CMP matrix and a gap weight of 40,
50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A set
of parameters often used is a Blossum 62 scoring matrix with a gap
open penalty of 12, a gap extend penalty of 4, and a frame shift
gap penalty of 5.
III. Methods of Sortagging and Processing Sortagged Cells
[0193] "Sortagging process" refers to a process in which at least
some entities (e.g., proteins, cells) become sortagged. In general,
a sortagging process comprises contacting an entity to be
sortagged, e.g., a eukaryotic cell, e.g., a mammalian cell, with a
transamidase and a sortase substrate under conditions in which a
sortase-catalyzed reaction can occur. In certain embodiments a
sortase reaction is performed under physiological conditions. In
general sortase-catalyzed conjugation may be performed by
contacting a transamidase, acyl donor (sortase substrate), and
nucleophilic acyl acceptor with one another under suitable
conditions to effect conjugation of the acyl donor to the acyl
acceptor. In embodiments of the present disclosure the nucleophilic
acyl acceptor may be a protein expressed by a mammalian cell.
Contacting the components with one another can be accomplished by
adding them to one body of fluid and/or in one reaction vessel, for
example, or otherwise placing the components in close proximity to
one another and allowing them to collide. The components in the
system may be mixed in a variety of manners, such as by oscillating
a vessel, if desired. The components may be added in any order to
the system. Conjugation may be performed in any convenient vessel
(e.g., tubes such as microfuge tubes, flask, dish), microtiter
plates (e.g., 96-well or 384-well plates), etc. The reaction
mixture may be maintained at any convenient temperature at which
the reaction can be performed. In some embodiments, the conjugation
is performed at a temperature ranging from about 3 or 4 degrees C.
to about 15 degrees C. In some embodiments, the conjugation is
performed at a temperature ranging from about 15 degrees C. to
about 50 degrees C. In some embodiments, the ligation is performed
at a temperature ranging from about 23 degrees C. to about 37
degrees C. In certain embodiments, the temperature is room
temperature (e.g., about 25 degrees C.). Any convenient volume and
component ratio may be utilized to conjugate a sortase substrate to
animal cells. In some embodiments, the acyl donor is present at a
concentration ranging from about 5 .mu.M to about 10 mM, about 10
.mu.M to about 5 mM, about 100 .mu.M to about 500 .mu.M, about 200
.mu.M to about 1 mM. In some embodiments the concentration of acyl
donor is at least 0.25 mM, at least 0.5 mM, or at least 1 mM. In
certain embodiments, the transamidase is present at a concentration
ranging from about 1 .mu.M to about 500 .mu.M, about 15 .mu.M to
about 150 .mu.M, about 150 .mu.M to about 250 .mu.M, about 250
.mu.M to about 500 .mu.M. In certain embodiments, the transamidase
is present at a concentration greater than 10 .mu.M, e.g., 11 .mu.M
to 20 .mu.M, 20 .mu.M to 30 .mu.M, 30 .mu.M to 50 .mu.M, 50 .mu.M
to 100 .mu.M. In some embodiments a transamidase is present at a
concentration greater than 10 .mu.M and is a wild type sortase,
e.g., a wild type sortase A, e.g., a wild type S. aureus SrtA, or a
variant thereof that has an activity between 0.5-fold and 5-fold
that of wild type S. aureus SrtA. In certain embodiments, the
transamidase is present at a concentration of about 10 .mu.M to 1
mM, about 1504 to about 1 mM, about 20 .mu.M to about 1 mM, about
25 .mu.M to about 1 mM, 30 .mu.M to about 1 mM, about 50 .mu.M to
about 1 mM, about 100 .mu.M to about 1 mM, or about 250 .mu.M to
about 1 mM. In certain embodiments, these concentrations apply to
wild type sortase A in particular, e.g., a wild type S. aureus
sortase A.
[0194] In certain embodiments conjugation is performed in a
reaction mixture comprising an aqueous medium. Water with an
appropriate buffer and/or salt content compatible with cell
viability may be used. One of ordinary skill in the art will be
familiar with a variety of buffers that could be used in accordance
with the present invention. In some embodiments, the aqueous medium
comprises calcium ions. For example, the aqueous medium may contain
between about 1.0 mM and about 50 mM calcium ions, e.g., from about
2 mM to about 25 mM calcium ions, e.g., from about 5 mM to about 15
mM calcium ions, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mM
calcium ions. In certain embodiments, the aqueous medium contains
between about 1 mM and about 5 mM calcium ions. In other
embodiments, the aqueous medium contains greater than 0.5 mM or
greater than 1 mM calcium ions. In certain embodiments, the aqueous
medium does not contain substances that bind to, or sequester
calcium ions or contains only trace amounts of such substances,
which would have negligible effects on the concentration of free
calcium ions. In certain embodiments, the aqueous medium does not
contain substances that precipitate calcium ions. In some
embodiments, the aqueous medium does not include phosphate ions. In
some embodiments, the aqueous medium does not include carbonate
ions. In some embodiments, the aqueous medium does not contain
chelating agents. For example, in some embodiments the aqueous
medium does not contain substances that chelate calcium ions, such
as EDTA or EGTA or contains only trace amounts of such substances,
which would have negligible effects on the concentration of free
calcium ions. In some embodiments, if the medium contains a
substance (other than a sortase) that binds to calcium ions, the
concentration of such substance is not sufficient to result in a
decrease in the catalytic activity of the sortase of more than 5%,
10%, 15%, 20%, or 25%. In some embodiments, the aqueous medium is
prepared without addition of calcium ions. In some embodiments the
concentration of calcium ions is below about 1.0 mM, e.g., below
about 0.5 mM, 0.25 mM, 0.1 mM, 0.05 mM, or lower. In some
embodiments, suitable ligation conditions comprise pH in the range
of 6 to 8.5, 6 to 8, 6 to 7.5, 6.5 to 8.5, 7 to 8.5, 7.5 to 8.5,
7.0 to 8.5, 7.3 to 7.8. It will be understood that the
afore-mentioned concentrations, ratios, and conditions are
exemplary and non-limiting. Higher or lower concentrations and/or
different conditions may be used in various embodiments.
[0195] When sortase substrates are conjugated to mammalian cells,
reaction conditions that are compatible with cell viability and, in
some embodiments, suitable to maintain normal cell function, are
used. Appropriate conditions within the range of conditions
described above may be used. For example, the temperature is within
a suitable range for mammalian cells, e.g., typically not more than
39 or 40 degrees, although higher temperatures, e.g., up to 45
degrees, may be used in certain embodiments. In some embodiments a
temperature between about 10 and 25 degrees C. may be used. In some
embodiments a temperature between about 25 and 37 degrees C. may be
used. In some embodiments a relatively low temperature, e.g.,
between about 4 and 10 degrees, may be used to reduce cellular
metabolism and/or internalization of cell surface proteins. In
embodiments in which non-mammalian cells are sortagged, reaction
conditions that are compatible with cell viability and, in some
embodiments, suitable to maintain normal cell function, are used.
Appropriate conditions within the range of conditions described
above may be used.
[0196] In some embodiments cells are at a concentration of between
about 10.sup.5 cells/ml and about 10.sup.12 cells/ml, e.g., between
about 10.sup.6 cells/ml and about 10.sup.11 cells/ml. A gentle
means of mixing may be used if desired such as gentle rocking. In
some embodiments mammalian cells are sortagged in a composition
comprising culture medium suitable for culturing the mammalian
cell(s). In some embodiments the culture medium is free or
essentially free of serum, plasma, and/or animal tissue or organ
extracts. In some embodiments the culture medium contains serum or
plasma. In some embodiments serum or plasma, if present, is from
the subject from whom the cells originated or to whom the cells are
to be administered. In some embodiments the culture medium is
chemically defined.
[0197] Parameters such as the concentration of sortase,
concentration of sortase substrate, number and/or concentration of
cells, ratio of sortase substrate and/or sortase to cells, aqueous
medium, and length of time of the reaction may be selected based on
a variety of factors, such as the activity of the particular
sortase, the nature of the sortase substrate (e.g., how readily it
serves as a substrate for sortase), the degree of conjugation
desired, etc. In some embodiments the sortase reaction may be
permitted to proceed for at least 15 minutes. In some embodiments
the sortase reaction may be permitted to proceed for more than 15
minutes. In some embodiments the sortase reaction may be permitted
to proceed for between about 30 minutes and about 24 hours, e.g.,
about 1-2, 2-4, 4-8, 8-12, 12-16, or 16-24 hours. In certain
embodiments samples may be removed from a reaction vessel and
tested for concentration of unconjugated sortase substrate or
reaction byproduct and/or level of agent conjugated to cells. The
reaction may be permitted to proceed until a desired endopoint is
reached. In certain embodiments the number of cells is up to about
10.sup.14 cells, e.g., about 10.sup.3, 10.sup.4, 10.sup.5,
10.sup.6, 10.sup.7, 10.sup.8, 10.sup.9, 10.sup.10, 10.sup.11,
10.sup.12, 10.sup.13 or 10.sup.14 cells, or any intervening range,
e.g., between about 10.sup.5 and about 10.sup.12 cells, between
about 10.sup.6 and about 10.sup.11 cells, between about 10.sup.7
and about 10.sup.10 cells. In certain embodiments sortase-mediated
modification of mammalian cells may be performed in 1-2 hours, or
less (e.g., between 15 and 30 minutes, between 30 and 60 minutes).
In some embodiments primary cells are obtained from a subject or
from a donor, modified in vitro using sortase, and at least some of
the modified cells are administered to the subject on the same day
as the cells were obtained, or the following day. In some
embodiments the complete procedure, from cell harvesting to
administration of modified cells, may take between 4-12 hours,
12-24 hours, or 24-48 hours. In certain embodiments cells that have
a poor survival rate or lose functional activity or alter their
phenotype when maintained in culture may be sortagged and
administered to a subject before losing viability or functional
activity or exhibiting altered phenotype.
[0198] In some embodiments a composition comprising one or more
living mammalian cells, sortase, and, in some embodiments, further
comprising a sortase substrate, is characterized in that at least
80%, 85%, 90%, 95%, 97%, 98%, 99%, or more of the cell(s) remain
viable in the composition for at least between 1-2 hours, 2-4
hours, 4-8 hours, 8-12 hours, or more, e.g., at least 12-24 hours.
In certain embodiments living mammalian cells modified using
sortase exhibit at most a 1%, 2%, 3%, 5%, 10%, 15%, 20%, or 25%
reduction in viability as compared to a suitable control. In some
embodiments, living mammalian cells modified using sortase retain
substantial functional activity. For example, in some embodiments
cells subjected to a sortagging process exhibit at most a 1%, 2%,
3%, 5%, 10%, 15%, 20%, or 25% reduction in at least one functional
activity as compared to a suitable control. In some embodiments,
living mammalian cells modified using sortase gain a new functional
activity or have an increased functional activity as compared with
a suitable control. For example, in some embodiments cells
subjected to a sortagging process may exhibit an increase of at
least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% in at
least one functional activity as compared to a suitable control. In
some embodiments cells subjected to a sortagging process may
exhibit at least a 2-fold, 5-fold, 10-fold, 20-fold, or more
increase in at least one functional activity as compared to a
suitable control. In some embodiments a new or increased functional
activity is conferred by or as a result of an agent conjugated to
the cells. In some embodiments a functional activity may be
measured in vitro. In some embodiments a functional activity or
change in a functional activity may be measured after
administration of cells to a subject. In some embodiments a
functional activity is binding activity, cytokine secretion,
cytotoxic activity, phagocytic activity, antigen presenting
activity, or costimulation activity. In some embodiments, living
mammalian cells modified using sortase may exhibit minimal or no
detectable non-specific alteration (e.g., oxidation, denaturation,
degradation) to cell surface proteins (other than those modified by
sortase-mediated conjugation) as compared with a suitable control.
In some embodiments a suitable control is cells of the same cell
type or subtype that have not been contacted with sortase. In some
embodiments a suitable control is cells of the same cell type or
subtype that have been contacted with sortase in the absence of a
sortase substrate. In some embodiments control cells may originate
from the same culture, cell line, or subject as the cells with
which they are compared. In some embodiments control cells not
contacted with sortase have been maintained under standard culture
conditions for that cell type or subtype (without sortase). In some
embodiments a suitable control refers to a value (e.g., for
viability or functional activity) measured for the cells prior to
contacting them with sortase. In some embodiments control cells not
modified using sortase have been incubated in a composition without
sortase for about the same length of time and under substantially
identical conditions as the sortase-modified cells with which they
are compared.
[0199] In some embodiments a sortase substrate comprising an agent
that has a functional activity is conjugated to living mammalian
cells with sortase. In some embodiments the agent retains at least
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or all of its
functional activity as compared to that of an unconjugated agent.
In some embodiments a functional activity is a catalytic activity,
an inhibitory activity, a binding activity, or a cytotoxic
activity. In some embodiments a functional activity is an ability
to stimulate a cell to survive, proliferate, become activated,
differentiate, migrate, produce or secrete one or more substances,
exhibit a phenotypic characteristic (e.g., expression of one or
more genes, cell surface marker phenotype), or attack a target
cell. In some embodiments a functional activity is an ability to
inhibit a cell from proliferating, becoming activated,
differentiating, migrating, producing or secreting one or more
substances, dying, altering a phenotypic characteristic (e.g.,
expression of one or more genes, cell surface marker phentopye), or
attacking a target cell. In some embodiments an agent confers an
ability to participate in a new cell-cell interaction. In some
embodiments an agent conjugated to a mammalian cell exerts an
autocrine effect. For example, the agent may bind to a cell surface
receptor expressed by a cell to which the agent is conjugated and
exert an effect on the cell. In some embodiments an agent exerts a
paracrine effect. For example, the agent may bind to a cell surface
receptor expressed by a cell located near the cell to which the
agent is conjugated and exert an effect on the cell. Binding of an
agent to a cell surface receptor may modulate a signaling pathway,
cause the cell to survive, differentiate, divide, migrate, maintain
or acquire a functional activity, etc.
[0200] In some embodiments mammalian cells are subjected to a
sortagging process, and at least some sortagged cells are then
separated from sortase, unconjugated sortase substrate, and/or
reaction byproduct. Separation may be performed using a variety of
different methods and may be based at least in part on size,
charge, affinity, hydrophobicity, hydrophilicity, and/or other
properties. In some embodiments sortase is immobilized by attaching
it to a support before or after being contacted with mammalian
cells. Immobilization may comprise contacting sortase or a
composition containing sortase with an affinity reagent, e.g., an
antibody, that binds to sortase, wherein the affinity reagent is
attached to a support. In some embodiments the sortase is tagged,
and the affinity reagent binds to the tag. In some embodiments the
support is in a column, and a composition comprising cells and
sortase is passed through the column. Sortase is retained by the
column whereas cells pass through. Cells may pass through the
column at a different rate to unconjugated agent, thereby achieving
separation. In some embodiments the agent comprises a tag that is
removed during sortagging as part of a reaction byproduct.
Unconjugated sortase substrate and/or reaction byproduct can be
removed by an affinity agent that binds to the tag. In some
embodiments sortase is immobilized before being contacted with
mammalian cells. For example, sortase may comprise a tag, e.g., a
6.times.-His tag, which may be used to immobilize the sortase to a
metal-ion containing resin or substrate. Mammalian cells are
incubated in the presence of the immobilized sortase and an agent
to be conjugated thereto. Cells can readily be separated from the
immobilized sortase. In some embodiments sortase is immobilized to
magnetic particles. It will be understood that magnetic particles
may be magnetisable and paramagnetic, e.g., superparamagnetic,
i.e., they may only magnetic in a magnetic field.
[0201] In some embodiments at least 5%, 10%, 15%, 20%, 25%, 30%,
40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more of
the mammalian cells in a population of cells subjected to a
sortagging process become conjugated with an agent. In some
embodiments a population of cells is processed after sortagging to
produce a composition in which at least 5%, 10%, 15%, 20%, 25%,
30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more
of the mammalian cells are conjugated with an agent. In some
embodiments mammalian cells that have been subjected to sortagging
may be separated into two, three, four, or more groups, e.g.,
between 2 and 10 groups, during or after being contacted with
sortase or may be subjected to selection. In some embodiments
separation or selection produces a population of cells that is
enriched in cells having a property that is desired and/or that is
at least partly depleted of cells having a property that is not
desired, as compared with a starting population prior to
separation.
[0202] In some embodiments cells that have been subjected to a
sortagging process are separated into two or more groups based at
least in part on the level of a moiety that has been conjugated
thereto by sortase. In some embodiments at least some cells that
have a moiety conjugated thereto at a level detectably greater than
a suitable control level are separated from cells that do not. In
some embodiments cells that exhibit at least a specified level of
moiety at their surface may be separated from cells that exhibit a
lower level or completely lack the moiety at their surface. Groups
may be defined based on the level of moiety using any suitable
classification system. In some embodiments cells are divided into
groups that are considered to exhibit low, intermediate, or high
levels. In some embodiments the 1%, 5%, 10%, 15%, 20%, 25%, 30%,
40%, 50%, 60%, 70%, 80%, or 90% of cells having the highest level
of moiety conjugated thereto are separated from the rest of the
cells in a population. A level may be an absolute amount, relative
amount, surface density, volume density, or other suitable
parameter. Suitable separation methods may be utilized so as to
produce a composition in which a selected degree of conjugation is
achieved. For example, in some embodiments a composition comprises
cells that have on average a specified level or range of levels of
moiety at their surface. A specified percentage may be a minimum
percentage, e.g., at least 10%, or a range, e.g., between 10% and
50%, between 50% and 90%, etc.
[0203] Any suitable method may be used to detect or measure the
level of an agent conjugated to cells or to separate cells into two
or more populations if desired. One of ordinary skill in the art
will be able to select an appropriate method taking into
consideration factors such as, for example, one or more physical,
chemical, or biological properties of the agent such as affinity
(e.g., binding affinity), charge, fluorescence, color, magnetic
properties, mass, enzymatic activity. In some embodiments a method
utilizes fluorescence, affinity, or both. For example, in some
embodiments an agent to be conjugated to cells comprises a
fluorescent moiety. Cells having the fluorescent moiety conjugated
thereto may be detected or measured using, e.g., flow cytometry or
immunofluorescence microscopy, and the level of agent may be
measured, if desired. Cells may be separated using fluorescence
activated cell sorting (FACS). In some embodiments an agent is
capable of binding to or being bound by a reagent (e.g. an
antibody) comprising a fluorescent label, and cells are contacted
with such a reagent under conditions suitable for binding to occur,
optionally followed by washing to remove non-specifically
associated reagent. Cells are then subjected to fluorescence
activated cell sorting. In some embodiments an affinity-based
method is used. For example, in some embodiments an agent to be
conjugated to cells comprises a tag. The tag may be detected using
a suitable reagent.
IV. Cells and Cell Culture
[0204] In general, any type of cells may be sortagged as described
herein or used as a source of cells to be sortagged. In some
embodiments cells comprise or consist of mammalian cells. In some
embodiments mammalian cells are primate cells (human cells or
non-human primate cells), rodent cells (e.g., mouse, rat, rabbit,
hamster cells), canine, feline, bovine, porcine, or other mammalian
cells. In some embodiments cells are avian cells. In some
embodiments cells are invertebrate animal cells. In some
embodiments cells are fungal (e.g., yeast or mold) or protozoal
cells.
[0205] A cell may be a primary cell, non-immortalized cell,
immortalized cell, normal cell, abnormal cell, tumor cell,
non-tumor cell, etc., in various embodiments. A cell may originate
from a particular tissue or organ of interest or may be of a
particular cell type. In some embodiments primary cells may be
freshly isolated from a subject. In some embodiments, cells are
maintained in culture and may be passaged or allowed to double once
or more following their isolation from a subject (e.g., between
1-5, 5-10, 10-20, 20-50, 50-80 passages or population doublings
times) prior to use in a method disclosed herein. In some
embodiments, cells have been passaged or permitted to double no
more than 1, 2, 5, 10, 20, or 50 times following isolation from a
subject before use in a method described herein. Cells "obtained
from a subject" may comprise originally isolated cells and/or
descendants thereof that arise during culture of the originally
isolated cells. In some embodiments cells are obtained from any
tissue or organ of interest. In some embodiments cells are obtained
from a fluid such as blood, sputum, lymph, mucus, saliva, urine,
blood, or lymph, from bone marrow, or lymphoid tissue (e.g., lymph
node, spleen). In some embodiments cells are obtained from
connective tissue, muscle tissue, adipose tissue, epithelial
tissue. In some embodiments cells are obtained from a tumor, site
of infection, site of inflammation or immune-mediated tissue
damage, or lymphoid tissue that receives lymph from such a site
(e.g., nearest draining lymph nodes).
[0206] In some embodiments a cell is a member of a cell line. In
some embodiments, a cell line is capable of indefinite
proliferation in culture (immortal; immortalized). An immortalized
cell line has acquired an essentially unlimited life span, i.e.,
the cell line appears to be capable of proliferating essentially
indefinitely. For purposes hereof, a cell line that has undergone
or is capable of undergoing at least 100 population doublings in
culture may be considered immortal. Numerous cell lines are known
in the art and may be used in various methods described herein.
Cell lines can be generated using methods known in the art or
obtained, e.g., from depositories or cell banks such as the
American Type Culture Collection (ATCC), Coriell Cell Repositories,
Deutsche Sammlung von Mikroorganismen and Zellkulturen (German
Collection of Microorganisms and Cell Cultures; DSMZ), European
Collection of Cell Cultures (ECACC), Japanese Collection of
Research Bioresources (JCRB), RIKEN, Cell Bank Australia, etc. The
paper and online catalogs of the afore-mentioned depositories and
cell banks are incorporated herein by reference.
[0207] In some embodiments a cell line, cell population, or cell
culture is derived from a single cell. In some embodiments, a cell
line, cell population, or cell culture is derived from multiple
cells. In some embodiments, cells of a cell line, cell population,
or cell culture are descended from a cell or cells originating from
a single sample (e.g., a sample obtained from a tumor) or
individual. If desired, cells may be tested to confirm whether they
are derived from an individual or a particular cell line by any of
a variety of methods known in the art such as DNA fingerprinting
(e.g., short tandem repeat (STR) analysis), single nucleotide
polymorphism (SNP) analysis (which may be performed using, e.g.,
SNP arrays (e.g., SNP chips) or sequencing), isoenzyme analysis,
human lymphocyte antigen (HLA) typing, chromosomal analysis,
karyotyping, morphology, etc.
[0208] An appropriate cell donor, cell source, or cell line may be
selected based on a variety of factors, such as the intended use of
the cells, number of cells desired, availability, etc. In some
embodiments, cells are obtained from an individual who is healthy
or is reasonably presumed to be healthy at the time the cells are
obtained. In some embodiments, cells are obtained from an
individual who does not have or is reasonably presumed not to have
one or more particular diseases, e.g., cancer, infection,
autoimmune disease, at the time the cells are obtained. In some
embodiments cells are obtained from an individual who does not have
a history of having a particular disease. In some embodiments cells
are obtained from an individual who has or has had a particular
disease. In some embodiments the disease is cancer, a disease
caused by a pathogen, or an autoimmune disease. In some embodiments
the subject exhibits resistance to a disease, e.g., a disease
caused by a pathogen. In some embodiments the subject is recovering
or has recovered from a disease. In some embodiments the subject is
in need of treatment of a disease, e.g., cancer, a disease caused
by a pathogen, an autoimmune disease, or a disease for which a
transplant is indicated. In some embodiments cells are obtained
from an individual who is histocompatible with a subject in need of
treatment of such a disease.
[0209] Cells used in a method described herein may have been
procured directly from a subject or procured indirectly, e.g., by
receiving the cells (or ancestors of the cells) through a chain of
one or more persons originating with a person who procured the
cells (or ancestors of the cells) directly from the subject, e.g.,
by performing a biopsy, blood draw, surgery, or other procedure on
the subject. In some embodiments at least some of the originally
isolated cells may undergo one or more rounds of cell division. In
some embodiments cells are obtained from a tissue biopsy such as an
excisional biopsy, incisional biopsy, or core biopsy; a fine needle
aspiration biopsy; a brushing; or a lavage. In some embodiments
cells are obtained from surgical or cellular samples from a subject
(e.g., tissue or cellular material harvested for purposes of
obtaining tissue or cells, excess or discarded tissue or cellular
material, etc.). A surgical sample may be obtained from an organ or
part of an organ that has been removed from a subject, e.g.,
because it is diseased or injured or enlarged. Methods of obtaining
samples and isolating cells from samples are well known in the art.
In some embodiments cells are obtained from a tissue sample. In
some embodiments cells are isolated from a tissue sample, by
dissociation, e.g., mechanical or enzymatic dissociation and may be
collected, e.g., by centrifugation, and washed, if desired. Cells
can be subjected to a variety of procedures to select or enrich for
cells of a desired cell type or having desired properties. In some
embodiments enrichment is performed at least in part based on
expression (which may be lack of expression) of one or more cell
surface markers using, e.g., FACS or affinity reagents. One can
select for against cells that express particular markers. In some
embodiments enrichment is performed at least in part by exposing
cells to an agent or combination of agents (e.g., cytokines) that
promote differentiation and/or expansion of one or more cell types.
In some embodiments a composition comprises at least 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90%, or more cells of a particular type
and/or expressing a particular marker or combination of markers. In
some embodiments at least some cells obtained from a subject and/or
descendants of such cells are stored, e.g., cryopreserved. Aliquots
may subsequently be thawed and used in one or more methods or
compositions described herein. In some embodiments cells may be
expanded in vitro prior to storage, after storage, or both.
[0210] In some embodiments cells may originate from any of the
three germ layers: ectoderm, mesoderm, and endoderm. In some
embodiments cells may originate from any biological tissue of the
four general classes of biological tissues: epithelial, muscular,
connective, and nervous tissues. In some embodiments cells comprise
epithelial cells. "Epithelium" refers to layers of cells that line
the cavities and surfaces of numerous structures in the body and is
the type of tissue from which many glands are at least partly
formed. Epithelial tissues include, for example, tissues found in
the gastrointestinal tract (e.g., esophagus, stomach, small
intestine, colon, rectum), liver, biliary tract, pancreas,
respiratory tract (e.g., nasal passages, pharynx, larynx, trachea,
bronchioles, bronchi, lungs, alveoli), oral cavity, skin, kidneys,
ovaries, breast, prostate, cervix, uterus, bladder, ureter, testes,
exocrine glands, endocrine glands, eye (e.g., retinal pigment
epithelium, corneal epithelium, conjunctiva), blood vessels
(vascular endothelim), lymph vessels (lymphatic endothelium), etc.
In some embodiments cells comprise or consist of muscle cells,
e.g., skeletal, smooth, or cardiac myocytes or myoblasts. In some
embodiments cells comprise or consist of connective tissue cells,
e.g., fibroblasts, adipocytes, cartilage cells (e.g., chondrocytes,
chondroblasts), bone cells (e.g., osteoblasts, osteoclasts). In
some embodiments cells comprise or consist of nervous system cells,
e.g., neural cells (e.g., neurons), glial cells (e.g., astrocytes,
oligodendrocytes, Schwann cells). In certain embodiments cells
comprise or consist of pancreatic beta cells, hepatocytes,
keratinocytes, or melanocytes.
[0211] In some embodiments cells comprise hematopoietic cells.
Hematopoietic cells include hematopoietic stem cells (HSCs), the
blood cells that give rise to all other blood cells, and cells of
the myeloid (e.g., monocytes and macrophages, neutrophils,
basophils, eosinophils, erythrocytes, megakaryocytes/platelets,
dendritic cells) and lymphoid (T cells, B cells, NK cells)
lineages. In some embodiments hematopoietic cells are obtained from
peripheral blood, e.g., by venipuncture. In some embodiments
hematopoietic cells are obtained by apheresis, a technique in which
the blood of an individual is passed through an apparatus that
separates one or more constituents and returns the remainder to the
circulation. For example, erythrocytapheresis may be used to obtain
red blood cells, leukapheresis may be used to obtain leukocytes
(white blood cells), plateletpheresis (also called thrombapheresis,
thrombocytapheresis) may be used to obtain platelets. Separation
may employ, e.g., centrifugation, elutriation, adsorbtion onto
resin (e.g., beads) with appropriate affinity, filtration, etc. In
some embodiments cells are obtained peripheral blood after
mobilization of such cells or their precursors, e.g., from bone
marrow. For example, in some embodiments bone marrow hematopoietic
stem cells (HSCs) are mobilized by, e.g., injections of
granulocyte-colony stimulating factor (G-CSF). In some embodiments
cells, e.g., HSCs or other hematopoietic cells, are isolated from
blood, e.g., peripheral blood or umbilical cord blood. In some
embodiments hematopoietic cells, e.g., HCSc, are isolated from
mobilized peripheral blood. HSCs may be expanded ex vivo and/or may
be differentiated ex vivo to yield lymphoid and/or myeloid cells of
one or more types.
[0212] In some embodiments cells comprise erythrocytes (red blood
cells) and/or committed progenitors thereof. Red blood cells (RBCs)
are typically abundant and readily accessible. In certain
embodiments RBCs may be of particular interest as vehicles for
delivery of therapeutic agents. In some embodiments, for example,
RBCs are sortagged with a therapeutic agent, e.g., a protein, a
chemotherapy drug, an anti-infective agent, etc., and administered
into the vascular system, e.g., intravenously.
[0213] In some embodiments cells comprise immune system cells. In
some embodiments an immune system cell is a lymphocyte, monocyte,
dendritic cell, macrophage, neutrophil, mast cell, eosinophil,
basophil, natural killer (NK) cell, or mast cell. In some
embodiments a lymphocyte is a cell of the B cell lineage or T cell
lineage. In some embodiments a B lymphocyte has rearranged its
heavy (H) chain gene. In some embodiments a B lymphocyte expresses
a membrane-bound antibody. In some embodiments a T cell expresses
an alpha beta (.alpha..beta.) T cell receptor (TCR). In some
embodiments a T cell expresses a gamma delta (.gamma..delta.) TCR.
In some embodiments a T cell is a member of a T cell subset, e.g.,
a cytotoxic T cell (also called killer T cell) or a helper T cell.
Cytotoxic T cells are typically positive for the cell surface
marker CD8. Helper T cells are typically positive for the cell
surface marker CD4. In some embodiments a cell is a CD4+CD8-T cell.
In some embodiments a cytotoxic cell is a CD4+ T cell, e.g., a
CD4+CD8-T cell. In some embodiments a cell is a CD4-CD8+ T cell. In
some embodiments a cell is a CD4-CD8-T cell. In some embodiments a
cell is a CD4+CD8+ T cell. In some embodiments a T cell is a
natural killer T (NKT) cell, e.g., an invariant NKT (iNKT) cell.
Natural killer T (NKT) cells are a subset of T cells that display
markers characteristic of both natural killer (NK) cells and T
cells. NKT cells recognize lipid or lipid-containing antigens
(e.g., glycolipids, lipopeptides) in the context of CD1 molecules.
NKT cells express an invariant TCR.alpha. chain rearrangement:
V.alpha.14J.alpha.18 in mice and V.alpha.24J.alpha.18 in humans,
which is associated with V.beta. chains of limited diversity, and
are sometimes referred to as canonical or invariant NKT (iNKT)
cells. Similar to conventional T cells, NKT cells develop from
CD4-CD8-thymic precursor T cells following the appropriate
signaling by CD1d. Human NKT cells can be stimulated and expanded
ex vivo by contacting them with .alpha.-galactosylceramide
(.alpha.-GalCer) and a variety of cytokines.
[0214] In some embodiments a T cell is a regulatory T cell (Treg),
e.g., a FoxP3+ regulatory T cell. In some embodiments a regulatory
T cell is a type 1 regulatory (Tr1) cell, which does not express
FoxP3. Tr1 cells typically secrete interleukin 10 (IL-10) and
transforming growth factor-.beta. (TGF-.beta.), e.g., in response
to antigenic stimulation. Tr1 cells are capable of dampening
autoimmunity and tissue inflammation partly through their secretion
of IL-10. In some embodiments a T cell is a follicular helper T
cell (T.sub.FH). T.sub.FH are antigen-experienced CD4+ T cells
found in the B cell follicles of secondary lymphoid organs such as
lymph nodes, spleens and Peyer's patches and may be identified by
their constitutive expression of the B cell follicle homing
receptor CXCR5. In some embodiments a T cell expresses and, in some
embodiments secretes, one or more cytokine(s) and/or has a
characteristic cell surface marker expression profile. For example,
in some embodiments a T cell has a T helper 1 (Th1), T helper 2
(Th2), or T helper 17 (Th17) cytokine secretion profile and/or cell
surface marker profile. Th1 cells are typically characterized by
production of interferon-.gamma. and TGF-beta. Th2 cells may
characteristically produce IL-4, IL-5, IL-6, IL-10, and IL-13. Th17
cells are typically characterized by production of
interleukin-17.
[0215] In some embodiments a lymphocyte is a naive cell (i.e., a
cell that has not encountered an antigen to which its B cell
receptor (BCR) or TCR binds and is not descended from a lymphocyte
that has encountered an antigen to which its BCR or TCR binds). In
some embodiments an immune system cell has encountered, in culture
or in vivo, an antigen to which its BCR or TCR binds, or is
descended from such a cell. In some embodiments an immune system
cell has been activated, in culture or in vivo. In some embodiments
an immune system cell is activated by exposure to an antigen
presenting cell (APC) that displays an antigen to which the cell's
TCR or BCR binds and/or by exposure to one or more cytokines. In
some embodiments an immune system cell having characteristics of
any of the afore-mentioned cell types may be generated at least in
part in vitro, e.g., by differentiation from a less differentiated
or naive cell. Protocols and reagents useful for generating such
cells are known in the art. Further information on varous immune
cell types may be found in, e.g., Zhu, J., et al., Differentiation
of effector CD4 T cell populations. Annu. Rev. Immunol., 28 (2010),
pp. 445-489; S. Crotty, Follicular helper CD4 T cells (TFH), Annu.
Rev. Immunol., 29 (2011), pp. 621-663.
[0216] In some embodiments a cell is a lymphokine-activated killer
cell (LAK). LAKs are a heterogeneous population of cells consisting
primarily of NK, NKT and T cells, which are generated in vitro by
culture of peripheral blood mononuclear cells (PBMCs) in IL-2 (see,
e.g., West, E J, et al., British Journal of Cancer (2011) 105,
787-795 and references therein, e.g., Grimm E A, et al. J Exp Med
155(6): 1823-1841). The predominant effector cells within LAKs are
believed to be NK cells, but LAKs may be more cytotoxic against
tumour cells, including otherwise NK-resistant targets, than
typical peripheral blood NK cells (Grimm et al, 1982). In some
embodiments LAKs or other immune system cells may be sortagged with
an agent comprising IL-2 or another cytokine or growth factor that
may exert an autocrine effect on the cells. Sortagging the cells
may provide an alternative to separately administering the cytokine
or growth factor (e.g., systemically), which may reduce unwanted
side effects that might otherwise be associated with such
administration.
[0217] In some embodiments a cell is an antigen presenting cell
(APC). An antigen-presenting cell (APC) is a cell that can process
and display foreign antigens in association with major
histocompatibility complex (MHC) molecules on its surface. T-cells
may recognize these complexes using their T-cell receptors (TCRs).
APCs may also display other molecules (costimulatory proteins) that
are required for activating naive T cells. In some embodiments APCs
express MHC class II molecules. Such APCs include dendritic cells,
macrophages, and B cells. In some embodiments APCs are capable of
stimulating CD4+ and CD8+ T cells. In some embodiments APCs
comprise professional APCs. In some embodiments professional APCs
are dendritic cells or macrophages. Dendritic cells (DCs) are a
class of white blood cells that occur in most tissues of the body,
particularly those in contact with the exterior such as the skin
(which contains a specialized dendritic cell type termed a
Langerhans cell) and mucosal surfaces, as well as in the blood.
During certain developmental stages DCs grow membranous projections
known as dendrites, from which the cell type gets its name. DCs
serve as a link between peripheral tissues and lymphoid organs and
play important roles in modulating the activity of other immune
system cells. Immature DCs sample the surrounding environment for
pathogens such as viruses and bacteria through pattern recognition
receptors (PRRs) such as toll-like receptors (TLRs). In response to
stimuli such as pathogen components or other danger signals,
inflammatory cytokines, and/or antigen-activated T cells, they
undergo maturation and migrate to the T cell area of lymph nodes or
spleen, where they display fragments of previously phagocytosed and
processed antigens at their cell surface using MHCII complexes. As
part of the maturation process, DCs upregulate cell-surface
receptors that act as co-receptors in T cell activation, such as
CD80 (B7-1), CD86 (B7-2), and/or CD40. DCs activate helper T cells
(Th cells) by presenting them with antigens derived from the
pathogen in the context of MHCII complexes, together with
non-antigen specific costimulators. Binding of CD4+ expressed at
the surface of Th cells to a non-polymorphic region of MHCII
enhances the physical interaction between DC and Th cells, allowing
potent stimulation of helper T cells that express TCR molecules
capable of binding the peptide. In addition, DCs have the capacity
to directly activate cytotoxic T cells and B-cells through
presentation of MHCII-peptide complexes and costimulators and are
also able to activate the innate arm of anti-tumor immunity, e.g.,
NK and NKT effector cells. DC stimulation promotes Th cell
proliferation, activation, and differentiation into effector Th
cells, memory Th cells, and regulatory Th cells. Effector Th cells
provide "help" to cytotoxic T cells, B cells, and macrophages by,
e.g., secreting cytokines that exert a variety of stimulatory
effects on these cell types. Th help promotes proliferation and
activation of cytotoxic T cells, stimulates B-cell proliferation,
induces B-cell antibody class switching, and stimulates antibody
production. Th stimulation also enhances the killing ability of
macrophages. Memory T cells play an important role in promoting the
rapid mounting of a specific, strong adaptive immune response upon
encountering an antigen to which a subject has previously been
exposed. Regulatory Th cells are believed to play an important role
in the self-limiting nature of the immune response. In some
embodiments, DCs capable of presenting a particular peptide
stimulate both the cell-mediated and humoral branches of the
adaptive immune response towards targets containing that peptide as
well as enhancing activity of the innate immune system. In some
embodiments DCs comprise immature DCs, which lack one or more
characteristics found in mature DCs present in tissues. For
example, immature DCs may lack dendrites and/or lack one or more
markers of mature DCs. In some embodiments immature DCs, e.g.,
immature human DCs, express and/or lack expression of CD83. In some
embodiments DCs, e.g., human DCs, comprise myeloid DCs. In some
embodiments DCs, e.g., human DCs, comprise plasmacytoid DCs. In
some embodiments DCs comprise plasmacytoid CD303+ DCs, myeloid
CD1c+ DCs, and/or myeloid CD141+ DCs. In some embodiments DCs,
e.g., immature DCs, are obtained from the blood or generated in
vitro from peripheral blood mononuclear cells (PBMCs). See, e.g.,
Tuyaerts, S., Cancer Immunol Immunother (2007) 56:1513-1537, for
discussion of DC generation, antigen loading methods and
immunomonitoring approaches that may be used.
[0218] In some embodiments immune system cells are generated or
expanded in vitro from, e.g., HSCs or myeloid lineage progenitor
cells.
[0219] In some embodiments a population of cells comprises immune
system cells of two or more types or subtypes, e.g., lymphocytes
and DCs, CD4+ T cells and CD8+ T cells, lymphocytes and NK cells,
etc. Any combination is encompassed. Two or more populations may be
individually isolated and subsequently combined. One or more of the
populations, or the combined population, may be sortagged.
[0220] In some embodiments cells comprise peripheral blood
mononuclear cells (PBMCs). As known in the art, PBMCs are
peripheral blood cells that have a round nucleus, such as
lymphocytes, monocytes, and NK cells. In some embodiments PBMCs are
sortagged as a mixed population comprising two or more distinct
cell types or subtypes distinguishable by size, morphology, cell
surface markers, and/or functional characteristics. In some
embodiments PBMCs are separated from other cells in a blood sample,
so that at at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more
of the cells are PBMCs. In some embodiments PBMCs are sortagged
without first separating the PBMCs into two or more types
distinguishable by size, morphology, cell surface markers, and/or
functional characteristics. In some embodiments PBMCs may be
separated into two or more distinct populations, wherein one or
more of the populations is enriched for one or more types of PBMC
or is depleted of one or more PBMC types. In some embodiments the
PBMCs are obtained from a subject to whom at least some of the
PBMCs or their descendants are to be administered after such cells
are sortagged ex vivo. PBMCs can be isolated using standard methods
known in the art, such as using Ficoll density gradient
centrifugation, which separates blood into a top layer containing
plasma and platelets, followed by a layer containing PBMCs, and a
bottom fraction containing polymorphonuclear cells (such as
neutrophils and eosinophils) and erythrocytes. The PBMC layer can
be removed, e.g., using a pipette, and sortagged. In some
embodiments a preparation containing PBMCs isolated from blood may
be further purified to remove residual red blood cells, e.g., prior
to sortagging or after sortagging. Red blood cell depletion from
blood or from a preparation containing PBMCs isolated from blood
may be performed by a variety of methods known in the art, such as
osmotic shock, filtration, density gradients such as
ficoll-hypaque, percoll and hydroxyethyl starch and immunoaffinity
with monoclonal antibodies such as CD34 coupled to magnetic beads.
In some embodiments, T cells may be isolated from PBMC. In some
embodiments a T cell subtype such as CD4+ or CD8+ T cells are
isolated from PBMC. In some embodiments, NK cells may be isolated
from PBMC. In some embodiments PBMCs are depleted of one or more of
cell such types. In some embodiments, memory T cells, e.g., central
memory T cells, are isolated from PBMC.
[0221] In some embodiments a cell is an artificial APC (aAPC).
Cellular aAPC may be derived, e.g., from primary or transformed
human or xenogeneic cells, e.g., fibroblasts or leukemia cells. In
some embodiments non-mammalian cells, such as insect (e.g., D.
melanogaster) cells may be used. Such cells may be engineered,
e.g., using retroviral or lentiviral transduction or other
approaches such as transposon systems, to cause them to express
molecules that provide TCR interaction, costimulatory, and adhesion
events involved in immune synapse formation, allowing them to
behave like naturally occurring APCs. Certain aAPCs are reviewed in
Kim, et al., Nature Biotechnology, 22(4): 403-410. In some
embodiments, such aAPCs maybe used or sortagged in accordance with
embodiments of the present invention. In some embodiments cells are
engineered to coexpress any one or more of the following: a low
affinity Fc receptor (e.g., CD32), a high affinity Fc receptor
(e.g., CD64), CD40, CD40L, CD70, CD80, CD83, CD86 (B7-2), ICOSL,
GITRL, CD137L (4-1BBL), CD252 (OX40L), B7-H3, ICAM-1, LFA-3, and/or
CD1. Some examples of genetically engineered aAPCs and methods of
making and using them are described in Maus, M. V. et al. Ex vivo
expansion of polyclonal and antigen-specific cytotoxic T
lymphocytes by artificial APCs expressing ligands for the T-cell
receptor, CD28 and 4-1BB. Nat. Biotechnol. 20, 143-148 (2002);
Thomas, A. K., et al. A cell-based artificial antigen-presenting
cell coated with anti-CD3 and CD28 antibodies enables rapid
expansion and long-term growth of CD4 T lymphocytes. Clin. Immunol.
105, 259-272 (2002). In some embodiments, cells may naturally
express one or more of the afore-mentioned molecules and,
optionally, are genetically engineered to express one or more
additional afore-mentioned molecules. In some embodiments cells
express at least two or all of the following: CD64, B7-2 (CD86),
and CD137 ligand (CD137L). In some embodiments cells express one or
more cytokines, e.g., IL-2, IL-12, or IL-15. In some embodiments,
cells express one or more membrane-bound cytokines, e.g.,
membrane-bound IL-15.
[0222] In some aspects, the present disclosure contemplates use of
sortagging to modify an aAPC or cell to be used as an aAPC by
conjugating any of a variety of agents to the cell surface. In some
embodiments, cells are sortagged with (CD137L), membrane-bound
IL-15, and/or other protein(s) that are normally expressed on the
cell surface and may act as ligands or interaction partners for
receptors or other molecules expressed on other cells, e.g., cells
to which an antigen is to be presented. Sortagging may be used in
some embodiments to attach one or more proteins to the cell surface
instead of or in addition to causing the cells to express such
proteins through use of genetic engineering. Sortagging may be used
in some embodiments to attach moieties that cannot be genetically
encoded, such as lipids or small molecules, optionally complexed or
attached to MHC proteins, CD1, or other proteins that normally
present antigens. Such moieties may be presented as antigens or
used for other purposes such as detection in vitro or in vivo.
[0223] In some embodiments, cells to be used as aAPC are sortagged
with an antigen of interest, resulting in aAPC that have an antigen
of interest attached to their cell surface. In some embodiments,
cells to be used as aAPC are genetically engineered to express an
antigen of interest at their cell surface, resulting in aAPC that
have an antigen of interest exposed at least in part on their
surface. The antigen of interest may be modified to include a
secretion signal sequence and transmembrane domain to cause it to
be expressed as a cell surface protein. In some embodiments, the
cells to be used as aAPC do not express HLA class I and/or HLA
class II molecules on their cell surface or at least do not express
HLA-A, HLA-B, HLA-DQ, and HLA-DR. In some aspects, a lack of such
major histocompatibility (MHC) antigens prevents immune responses
that may otherwise be directed against an aAPC that expresses such
MHC antigens if the aAPC is cultured with non-allogeneic immune
cells and/or introduced into a non-allogeneic subject. In some
embodiments cells to be used as aAPCs are engineered to express an
HLA class I molecule, HLA class II molecule, or both. In some
embodiments the cells do not otherwise express HLA class I, HLA
class II, or both. In some embodiments, cells to be used as aAPC
are contacted with a soluble antigen, e.g., in vitro. In some
embodiments, the cells take up, process, and display the antigen or
fragments thereof (e.g., peptides) on their surface in association
with an HLA class I and/or class II molecule. In some embodiments,
cells to be used as aAPC are sortagged with an antigen presenting
molecule (APM). For example, cells may be sortagged with a molecule
comprising at least a portion of an MHC protein that is capable of
binding to an antigen. In some embodiments the antigen presenting
molecule has been contacted or is contacted (e.g., in vitro) with a
peptide or other antigen such that the peptide or other antigen is
bound to the APM. In some embodiments an APM comprises at least a
portion of an MHC protein that is capable of binding to an antigen,
e.g., an MHC multimer or an HLA-Ig fusion protein (Oelke, 2003,
full citation below).
[0224] aAPCs may be used for a variety of purposes, e.g., in the
stimulation (activation and/or expansion) and/or positive and
negative modulation of cellular immune responses, e.g., of
lymphocytes (T and/or B cells), NK cells, or other cells. aAPCs may
be used in vitro, e.g., to activate and expand immune cells that
are normally stimulated by or require antigen presentation (e.g.,
engagement of the TCR by antigen in an appropriate context, e.g.,
with provision of costimulatory signals by other cells) for
maturation, proliferation, or acquisition of effector functions.
aAPCs may be used, for example, for the in vitro stimulation of
immune system cells that are to be administered to a subject. In
some embodiments a composition comprises aAPC and immune cells to
be stimulated. In some embodiments the composition further
comprises at least one cytokine (e.g., IL-2, IL-7, IL-12, IL-15,
IL-21), anti-CD3 monoclonal antibody such as OKT3, and/or anti-CD28
monoclonal antibody. In some embodiments the composition does not
comprise anti-CD3 and/or anti-CD28 antibodies. In some embodiments,
aAPCs are cultured in the presence of anti-CD3 monoclonal antibody
such as OKT3, and/or anti-CD28 monoclonal antibody prior to
contacting them with immune cells to be stimulated and are
subsequently cultured in the presence of cells to be stimulated,
optionally in the absence of anti-CD3 and/or anti-CD28 antibodies.
In some embodiments, the culture does not comprise further comprise
feeder cells (other than the aAPCs and/or such cells as may be
incidentally present in a population of cells to be stimulated by
the aAPCs and may act as feeder cells, i.e., cells are not added
specifically to act as feeder cells). In some embodiments, aAPC
that express an antigen to which a BCR, TCR, or CAR binds are used
to stimulate the cells to cause them to proliferate and/or become
activated in vitro. In some embodiments aAPC are used in vivo. For
example, in some embodiments aAPC are administered to a subject,
e.g., to stimulate endogenous immune system cells or immune system
cells administered to the subject (or their descendants).
[0225] In some embodiments cells comprise phagocytic cells.
Phagocytic cells may be professional or non-professional
phagocytes. Professional phagocytes include macrophages, monocytes,
dendritic cells, mast cells, and neutrophils.
[0226] In some embodiments cells comprise adult stem cells. Adult
stem cells are undifferentiated cells that exist in post-natal,
e.g., adult, organisms and are capable of giving rise to multiple
different cell types. Adult stem cells may be characterized by
self-renewal (the ability to go through numerous cycles of cell
division giving rise to daughter cells at least one of which
maintains an undifferentiated state) and multipotency or
multidifferentiation potential, i.e., the ability to give rise to
descendants of multiple distinct cell types. An adult stem cell may
have multlineage potential and/or may be capable of generating all
the cell types of the organ or tissue from which it originates,
potentially regenerating an entire organ from a few cells. Adult
stem cells may undergo symmetric division, which gives rise to two
identical daughter cells, both endowed with stem cell properties or
asymmetric division, which produces one stem cell and a progenitor
cell with more limited self-renewal potential. Progenitor cells can
go through at least one round of cell division, typically several
rounds of cell division, before differentiating into a mature cell.
Examples of adult stem cells include, e.g., hematopoietic stem
cells, neural stem cells, endothelial stem cells, intestinal stem
cells, mammary stem cells, mesenchymal stem cells, neural crest
stem cells. Adult stem cells may be further differentiated using
appropriate protocols known in the art. In some embodiments cells
comprise or consist of gametes (egg or sperm cells) or germ cells.
In some embodiments cells comprise pluripotent cells, e.g.,
embryonic stem cells. In some embodiments mesenchymal stem cells
(MSCs) comprise adherent non-hematopoietic bone marrow-derived stem
cells. In some embodiments MSC may be characterized by: plastic
adherence, maintenance of tri-lineage (osteogenic, adipocytic, and
chondroblastic differentiation potential after in vitro
propagation), and lack of the hematopoietic markers CD45, CD34,
CD14, CD11b, CD79-a, CD19, and HLA-DR, and simultaneous expression
of the surface molecules CD73, CD90, and CD105 on at least 95% of
the population (Dominici M et al., (2006) Minimal criteria for
defining multipotent mesenchymal stromal cells. The International
Society for Cellular Therapy position statement. Cytotherapy
8:315-317).
[0227] In some embodiments cells may be generated in vitro by
reprogramming a somatic cell to a less differentiated state or by
reprogramming a somatic cell from a first differentiated state to a
second differentiated state (sometimes termed
"transdifferentiation"). In some embodiments reprogramming
comprises reprogramming a cell to a multipotent or pluripotent
state. In some embodiments a reprogrammed cell is an induced
pluripotent stem (iPS) cell. In some embodiments an iPS cell may be
generated by causing the cell to express or contain one or more
genetic factors ("reprogramming factors"). Suitable combinations of
reprogramming factors are known in the art. In some embodiments
reprogramming factors include one or more factor selected from
Oct4, Sox2, Klf4, Nanog, Lin28, and c-Myc. Examples of suitable
combinations include, e.g., (1) Oct4, Klf4, Sox2, and optionally
c-Myc; (2) Oct4, Sox2, Nanog and Lin28; (3) Oct4, Esrrb, Nanog; (4)
Sox2, Sal14, Nanog; (5) Lin28, Sal14, Esrrb, Nanog; (6) Lin28,
Sal14, Esrrb, Nanog. Nanog may be replaced by Dppa2. A variety of
small molecules that enhance reprogramming and/or substitute for or
induce expression of one or more reprogramming factors are known
and may be used in various embodiments. Such molecules may, e.g.,
activate or inhibit one or more signaling pathways or may cause
alterations in chromatin structure. In some embodiments a somatic
cell to be reprogrammed is a fibroblast, keratinocyte, or
hematopoietic cell. In some embodiments a somatic cell is
reprogrammed using Oct4 and at least one small molecule. In some
embodiments nucleic acid(s) encoding one or more reprogramming
factors operably linked to regulatory elements capable of directing
transcription is introduced into cells using a viral vector, e.g.,
a retroviral vector. A copy of the coding sequence(s) may integrate
into the genome. In some embodiments such nucleic acid is
subsequently at least partially excised from reprogrammed cells. In
some embodiments reprogramming is performed using a method that
does not involve altering the genome of a cell. For example,
translatable RNA encoding one or more reprogramming factors may be
introduced into the cell and/or a cell may be contacted with one or
more small molecules that promote reprogramming. In some
embodiments translatable RNA may be synthetic modified RNA. Methods
that may be used for reprogramming using synthetic modified RNA are
described, e.g., in PCT/US2011/032679 (WO2011130624) and/or in
Mandal P K, Rossi D J. Reprogramming human fibroblasts to
pluripotency using modified mRNA. Nat Protoc. 2013; 8(3):568-82. In
some embodiments, reprogramming mammalian somatic cells may
comprise stressing the cells by, e.g., transient exposure to
chemical or physical stimuli such as low-pH conditions (e.g., about
pH 5.7). The cells may thereby be stimulated to undergo
stimulus-triggered acquisition of pluripotency (STAP), and cell
lines comprising pluripotent cells may be derived under appropriate
conditions (see, e.g., Obokata, H., et al., Stimulus-triggered fate
conversion of somatic cells into pluripotency. Nature, 2014; 505
(7485): 641-647; and Obokata, H., et al., Bidirectional
developmental potential in reprogrammed cells with acquired
pluripotency. Nature, 2014; 505 (7485): 676-680). In some
embodiments lymphocytes are reprogrammed via STAP. In some
embodiments the reprogramming does not entail introducing exogenous
nucleic acids or proteins into the cells. In some embodiments a
cell that has been reprogrammed to a less differentiated state,
e.g., to pluripotency is induced to differentiate into one or more
selected cell types or along one or more selected cell lineages.
For example, a pluripotent cell may be induced to have properties
of an adult stem cell. Such cells may be further differentiated
using appropriate protocols known in the art. Adult stem cells or
cells differentiated therefrom may be sortagged as described
herein. In some embodiments, for example, pluripotent cells
generated from somatic cells may be reprogrammed to hematopoietic
cells, e.g., immune system cells, e.g., T cells, B cells, NK
cells.
[0228] Cells may be cultured in any suitable cell culture vessel or
using any suitable cell culture system. In some embodiments plates
(e.g., multiwell plates) or flasks, e.g., conventional tissue
culture plasticware, may be used. A cell culture system may
comprise means for regulating oxygen and/or carbon dioxide
concentration, pH, or other cell culture relevant parameters. In
some embodiments a cell culture system, sometimes termed a cell
bioreactor, that provides mechanical rocking or stirring or pumping
(e.g., sparging) to perfuse media with gas or that provides
continuous or intermittent media flow or exchange may be used. The
use of such systems may enhance cell expansion, which may result in
higher cell densities than typically attained using conventional
plasticware. A variety of cell culture systems (e.g., hollow fiber
bioreactors, stirred tank bioreactors, bags, etc.) useful for
culturing cells are known in the art. In some embodiments a
gas-permeable bag or vessel comprising a gas-permeable membrane,
e.g., a silicone membrane, may be used. In some embodiments
Vuelife.TM. bags (Cellgenix, Freiburg, Germany), a WAVE
Bioreactor.TM. system (GE Health, Uppsala, Sweden), BIOSTAT.RTM.
CultiBag RM system (Sartorius Stedim Biotech, Gottingen, Germany),
or G-Rex system (Wilson Wolf Manufacturing, New Brighton, Minn.),
or system employing similar technology, may be used. In some
embodiments, for example, a cell culture system may comprise a cell
culture vessel comprising a gas-permeable cultureware flask in
which O.sub.2 and CO.sub.2 are exchanged across a gas-permeable
membrane at the base of the flask. In some embodiments a cell
culture vessel may have a volume or recommended media volume of,
e.g., up to 5 ml, 10 ml, 15 ml, 20 ml, 25 ml, 30 ml, 40 ml, 50 ml,
75 ml, 100 ml, 200 ml, 300 ml, 400 ml, 500 ml, 750 ml, or 1,000 ml.
In some embodiments a cell culture vessel may have a volume or
recommended media volume of more 1 liter (1), e.g., up to 1.5, 2.0,
2.5, 3.0, 4.5, 5.0 liters, or more. In some embodiments a selected
number of cells may be cultured produced in a single culture vessel
or system or in some embodiments using multiple culture vessels or
systems. In some embodiments a selected number of cells may be
cultured produced without need for stirring. In some embodiments a
selected number of cells may be cultured produced without need for
medium change, or with only 1, 2, 3 medium changes. In some
embodiments a selected number of cells may be, e.g., up to about
10.sup.14 cells, e.g., about 10.sup.3, 10.sup.4, 10.sup.5,
10.sup.6, 10.sup.7, 10.sup.8, 10.sup.9, 10.sup.10, 10.sup.11,
10.sup.12, 10.sup.13 or 10.sup.14 cells, or any intervening range,
e.g., between about 10.sup.5 and about 10.sup.12 cells, between
about 10.sup.6 and about 10.sup.11 cells, between about 10.sup.7
and about 10.sup.10 cells. In some embodiments between about
10.sup.5-10.sup.8 cells and about 10.sup.11-10.sup.13 cells are
cultured or produced. In some embodiments a system allows for
medium changes without removing a lid. In some embodiments cell
culture is performed under aseptic conditions. In some embodiments
cells may be cultured on microcarriers.
[0229] In some embodiments cells are obtained, e.g., from a
subject, and expanded in vitro. Cell "expansion" refers to an
increase in the number of cells. In some embodiments cells are
cultured for between about 48 hours and about 12 weeks, e.g.,
between 2 and 6 weeks. In some embodiments the number of cells is
increased by a factor of at least 2, e.g., between 2 and 100,
between 100 and 500, between 500 and 1,000, between 1,000 and
5,000, between 5,000 and 10,000, between 10,000 and 50,000, between
50,000 and 100,000, or more, relative to the number of cells in an
initial sample or culture. In some embodiments expanded cells
substantially retain or exhibit a particular phenotype, functional
activity, or cell surface marker profile of interest. In some
embodiments an expanded culture may be subjected to sorting or
separating or selection to enrich for cells having a selected
phenotype, functional activity, or cell surface marker profile. In
some embodiments an expanded culture may be characterized for a
selected phenotype, functional activity, or cell surface marker
profile.
[0230] Protocols and useful reagents and culture systems suitable
for culturing and/or expanding a wide variety of cell types are
known in the art. In some embodiments a cell culture medium, cell
culture system, sortase preparation, sortase substrate, cell
culture process, or sortagging process complies with Good
Manufacturing Practices (GMP). In some embodiments a sortase
preparation that is free or substantially free or essentially free
of endotoxin may be used. In some aspects, the present disclosure
provides GMP-compliant compositions comprising one or more cells
that have an agent conjugated by sortase to a non-genetically
engineered endogenous polypeptide expressed by the cell. In some
embodiments cell culture, sortagging, or both, are performed under
conditions appropriate to permit subsequent introduction of the
sortagged cells into a human subject. In some embodiments a cell
composition may be subjected to any one or more tests used in the
art to assess suitability for administration to humans and/or for
veterinary purposes. In some embodiments a cell composition
satisifies any one or more criteria used in the art to assess
suitability for administration to humans and/or for veterinary
purposes. In some embodiments culture conditions include use of
culture medium that is free or essentially free of serum, plasma,
and/or cell and tissue-derived substances extracts. In some
embodiments culture conditions may include absence of feeder cells.
In some embodiments, if one or more such substance(s) or feeder
cells is used, they have been appropriately tested to confirm that
they are free of human pathogens and/or substances that would
render them potentially unsafe or unsuitable for administration to
human subjects. In some embodiments, if one or more such
substance(s) or feeder cells is used, it is obtained from the same
subject as that from which the cells are obtained or a subject to
whom the cells are to be administered. In some embodiments one or
more recombinantly produced proteins may be used. In some
embodiments the composition comprises a chemically defined culture
medium. Suitable culture medium may be obtained from a variety of
commercial suppliers, e.g., Stemcell Technologies Inc. (Vancouver,
BC, Canada), Life Technologies, Inc. (Carlsbad, Calif.), Lonza
(Basel, Switzerland). For example, in some embodiments StemSpan
medium may be used, e.g., to culture hematopoietic cells. In some
embodiments AIM V.RTM. Medium may be used, e.g., to culture immune
system cells such as lymphocytes, monocytes, dendritic cells,
natural killer cells, PBMC, macrophages, etc. In some embodiments
GIBCO OpTmizer.TM. CTS.TM. T-Cell Expansion Serum Free Medium (Life
Technologies) is used, e.g., for culture of human T lymphocytes. In
some embodiments X-VIVO.TM. medium (Lonza), e.g., X-VIVO.TM. 10,
X-VIVO.TM. 15, or X-VIVO.TM. 20 is used, e.g., to culture immune
system cells such as lymphocytes, monocytes, dendritic cells,
natural killer cells, PBMC, macrophages, etc. In some embodiments
RPMI, DME, or DMEM may be used. One of ordinary skill in the art
will be aware of suitable culture media for cell types of
interest.
[0231] As used herein, a composition may be considered "free" of a
particular material or substance if the material or substance is
not deliberately added to or known to be present in the composition
and/or is undetectable using standard methods used in the art for
detection of such material or substance and/or if the composition
has been prepared under conditions accepted in the art as
sufficient to achieve absence of the material or substance. In some
embodiments a composition may be substantially free or essentially
free of any one or more materials or substances. In some
embodiments "essentially free" refers to a concentration of no more
than 0.1%. 0.05%, 0.01%, 0.005%, 0.001%, 0.0005% of such material
or substance by weight (e.g., dry weight), volume, or by moles. In
some embodiments "substantially free" refers to 1% or less, e.g.,
0.5% or less, e.g., 0.2% or less of such material or substance by
weight (e.g., dry weight), volume, or by moles. In some embodiments
a composition is considered substantially free of a material or
substance, e.g., an adjuvant, if the component or substance is not
detectable using a standard detection method used in the art for
detecting such material or substance. In some embodiments a
composition is prepared without deliberately including a substance,
e.g., an adjuvant. In some embodiments a composition is prepared
without deliberately including an adjuvant in an amount that would
be effective to enhance an immune response when the composition is
contacted with cells in vitro or in vivo.
[0232] In some embodiments cells cultured or sortagged in the
composition satisfy regulatory requirements for administration to a
human subject. In some embodiments cells cultured or sortagged in
the composition satisfy regulatory requirements of a government
agency such as the US Food and Drug Administration, European
Medicines Evaluation Agency, or a similar agency responsible for
evaluating the safety of therapeutic agents prior to their
administration to humans or being placed on the market for
administration to humans. In some embodiments cells are washed or
otherwise processed to remove media components after sortagging,
e.g., prior to administration to a subject. In some embodiments a
composition is tested and determined to be free of pathogens that
may infect humans. In some embodiments a test may utilize PCR or a
biological assay for presence of a particular pathogen.
[0233] Methods for culturing, expanding, and, in some embodiments,
activating, cells of various types are known in the art. For
example, T cells may be expanded and activated using antibodies
that bind to CD3 (e.g., Muromonab-CD, also known as Orthoclone
OKT3, "OKT3"), optionally in combination with one or more cytokines
such as IL-2. In some embodiments immune system cells may be
exposed to costimulatory signals provided by soluble,
surface-bound, or cell-bound (e.g., APC-bound) costimulatory
molecules, such as CD28, for example. In some embodiments immune
system cells may be contacted in vitro with an antigen or epitope,
optionally in association with an MHC protein. Hematopoietic stem
cells may be culturing and/or expanded as described, e.g., in US
Pat. Pub. Nos. 20110117061; 20110136230; and/or 20110196343. Such
cells may be differentiated along various hematopoietic lineages if
desired.
[0234] Examples of systems and/or protocols that may in various
embodiments be used to culture, and, in some embodiments, expand
and/or activate, cells are described in PCT/US2002/028161
(WO/2003/024989); PCT/US2004/001349 (WO/2004/065590);
PCT/US2008/062687 (WO/2009/136907); PCT/US2009/049944
(WO/2010/006055); PCT/US2010/046505 (WO/2011/028531);
PCT/NL2006/000319 (WO/2007/001173); PCT/US2010/061706
(WO/2011/079165); PCT/EP2012/063034 (WO/2013/007574);
PCT/SE2010/050333 (WO/2010/110734); Digiusto, D L and Cooper, L J
N, Cytotherapy. 2007; 9(7):613-29; Sutlu, T., et al., Cytotherapy.
2010 December; 12(8):1044-55; Spanholtz J, et al., PLoS One. 2011;
6(6):e20740. doi: 10.1371/journal.pone.0020740, Levine, B. et al.,
J Hematother. (1998) 7(5):437-48; Tumaini, Cytotherapy. 2013;
15(11):1406-15, and references in any of the foregoing).
[0235] In some embodiments cells are separated into two or more
groups prior to sortagging, and only one or some (but not all) of
the groups are subjected to a sortagging process. For example, in
some embodiments cells of a particular cell type may be selected
for sortagging from a starting population comprising cells of
multiple types. Cells of other types that may be present in the
starting population may, for example, be used for other purposes,
stored (e.g., cryopreserved), discarded, or subsequently combined
with sortagged cells. Cells may be selected based on any one or
more properties. A property may be any phenotypic characteristic of
a cell, cell type, or cell state, e.g., any characteristic that may
be observed or detected, or any combination of such
characteristics. Examples include, e.g., morphologic
characteristics; physical, biochemical, or physiological
properties; biological behavior, the presence, absence, or level of
gene expression products such as RNA or proteins. In some
embodiments a phenotypic characteristic is quantifiable. In some
embodiments cells may be separated based on size, light scattering,
density, binding affinity for one or more substances, expression of
at least one gene as assessed, e.g., by the level of a gene product
of the gene.
[0236] Any suitable separation method(s) may be used. Multiple
steps of selection or separation may be used. In some embodiments
selection comprises at least one positive selection, wherein
desired cells are retained or enriched for based on one or more
properties of the cells of interest (wanted cells; desired cells),
e.g., using a binding agent that binds to cells of interest. In
some embodiments selection may comprise at least one negative
selection, wherein cells that are not of interest (unwanted cells;
undesired cells) are removed or depleted based on one or more
properties of the unwanted cells (e.g., using a binding agent that
binds to unwanted cells). Examples of useful separation methods
include centrifugation, elutriation, contacting with an affinity
resin (e.g., beads) that retains or removes unwanted cells, flow
cytometry, fluorescence activated cell sorting (e.g., after
contacting the cells with appropriately labeled reagents that bind
to cell surface markers characteristic of cells whose removal or
retention is desired).
[0237] In some embodiments cells having a particular cell surface
marker expression profile are selected or removed. A cell surface
marker expression profile may comprise presence, absence, and/or
level of any one or more cell surface markers. Cell surface marker
expression profiles characteristic of many different cell types or
cell subsets having various functional characteristics of interest
are known in the art. In some embodiments a cell surface marker
comprises a cluster of differentation (CD) molecule. In some
embodiments an antibody-based enrichment procedure is used, which
may be combined with columns, magnet-based separation, and/or
centrifugation. In some embodiments magnetic particles (particles,
whether or not magnetic, are sometimes termed "beads") may be used.
Examples of such particles include those known as Dyabeads (Life
Technologies, Carlsbad, Calif.), MACS microbeads (Miltenyi Biotech,
Auburn, Calif.). In some embodiments, cells in a single-cell
suspension are magnetically labeled with beads that have an
appropriate binding agent (e.g., an antibody) attached thereto. The
sample is applied to a column placed in a magnetic separator. The
unlabeled cells pass through while the magnetically labeled cells
are retained within the column. The flow-through can be collected
as the unlabeled cell fraction. The column may be washed and
removed from the separator, and the magnetically labeled cells
eluted from the column. Thus both labeled and unlabeled cells can
be isolated if desired. In some embodiments beads may have any
appropriate dimensions, e.g., diameter, volume. In some embodiments
desired cells are obtained without using columns, without using
magnets, and/or without labeling desired cells with a binding
reaget such as an antibody. For example, whole blood may be
contacted with antibody complexes that comprise two or more
antibodies to antigen(s) expressed at the surface of red blood
cells but not expressed at the surface of desired cells and one or
more antibodies to antigen(s) expressed at the surface of cells to
be removed. In some embodiments a tetrameric antibody complex may
be used. The antibody complexes crosslink undesired cells to red
blood cells present in the whole blood, resulting in complexes
(sometimes termed immunorosettes) that can be removed by
centrifugation, e.g., over an appropriate density medium (e.g.,
Ficoll.TM.). Centrifugation may be used to pellet the
immunorosettes, thereby removing the unwanted cells along with red
blood cells and leaving desired cells. The purified cells are
present as an enriched population at the interface between the
plasma and the buoyant density medium. Reagents suitable for
performing such separation are commercially available. For example,
RosetteSep.RTM. reagents or kits may be used (Stemcell
Technologies) may be used. This technology utilizes tetrameric
antibody complexes that crosslink unwanted cells to multiple red
blood cells already present in the sample, forming immunorosettes.
When centrifuged over the appropriate density medium (e.g. Ficoll),
the unwanted (rosetted) cells pellet along with the red blood
cells, leaving the desired cells untouched and highly enriched at
the density medium: plasma interface. RosetteSep.TM. may be used on
its own with standard density gradient centrifugation or with a
specialized cell processing tube (SepMate.TM.). Suitable RBC
antigens include, e.g., glycophorin, e.g., glycophorin A. In some
embodiments isolation of desired cells from a mixed sample is
performed using a procedure that takes no more than 30 minutes or
no more than 60 minutes. In some embodiments separation may be
performed at least in part using an automated system. For example,
RoboSep.TM. (Stemcell Technologies) is an instrument that provides
for automation of immunomagnetic cell separation performing the
steps necessary to magnetically label and separate virtually cells
of a selected cell type by positive or negative selection. In some
embodiments a selection procedure may comprise culturing cells
under selective conditions, wherein the selective conditions kill
or inhibit proliferation of cells having certain characteristics
that are not desired. In some embodiments selective conditions
comprise culturing a cell population comprising multiple cell types
in the absence of one or more factors required for survival or
proliferation of unwanted cells.
[0238] In some embodiments mammalian cells may be assessed for
immunocomptibility with a subject to whom they are to be
administered. Cells may be deemed immunocompatible if they are
unlikely to provoke a significant immune response in a subject to
whom they are administered, e.g., an immune response that would
materially reduce the viability and/or functional activity of the
cells or produce excessive symptoms in a subject. In some
embodiments immunocompatiblity comprises histocompatibility.
Histocompatibility may be assessed using any suitable method known
in the art. In some embodiments histocompatibility testing
comprises determining whether a potential recipient has antibodies
to HLA antigens expressed by cells to be administered and/or
whether a potential recipient has an HLA genotype that differs from
that of the cells to a sufficient extent as to be deemed
incompatible (e.g., likely to be or at risk of being subject to
attack by the recipient's immune system) as reasonably determined
by one of ordinary skill in the art. In some embodiments cells to
be administered to a human subject are tested to determine their
HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and/or HLA-DR genotype and/or
a sample from a subject is tested to determine whether it contains
antibodies to MHC Class I and/or MHC Class II antigens of cells to
be administered to the subject. In some embodiments cells are
deemed histocompatible for administration to a subject if they have
at least the same HLA-A, HLA-B, and HLA-DR alleles as the subject's
cells and/or if the subject's blood does not harbor antibodies to
MHC molecules expressed by the cells. Methods of testing for
histocompatibility include, e.g., screening for preformed
alloreactive antibodies using complement-dependent cytotoxicity
assays (CDC) which detects complement-activating IgG1/3 and IgM
antibodies using a panel of HLA-typed lymphocytes to identify
reactive antibodies, solid-phase methods such as ELISA, and flow
cytometry-based techniques (using classical flow cytometry or
Luminex). For example, Luminex-based antibody screening technology
uses purified HLA antigens immobilized on a panel of microbeads.
These HLA molecules on the microbeads are targets for HLA-specific
antibodies in a given sample. Anti-HLA antibodies of the IgG
isotypes which are bound to the microbeads are detected by a
secondary IgG-specific antibody which may conjugated with a label
such as R-phycoerythrin (PE). Nucleic acid-based (e.g., DNA-based)
tissue typing may be performed using, e.g., sequence-specific
primers and/or sequence-specific probes (see, e.g., Dunckley H. HLA
typing by SSO and SSP methods. Methods Mol Biol. 2012; 882:9-25).
Primers may be used to amplify relevant sequences, e.g., using PCR.
Probes may be attached to oligonucleotide arrays or beads. In some
embodiments sequencing, e.g., high throughput sequencing, may be
used for genotyping. In some embodiments techniques generating
low-resolution (i.e. 2-digits HLA nomenclature) results or
high-resolution (i.e. at least 4-digits HLA nomenclature) results
may be used. Intermediate HLA resolution comprises a 2-digits HLA
nomenclature with supplemental characters to define groups of HLA
alleles. In general, an HLA typing resolution and/or degree of HLA
matching at a level relevant for different clinical applications
may be used.
[0239] In some embodiments immunocompatible red blood cells are of
the same blood group as an individual to whom such cells are to be
administered (e.g., at least with respect to the ABO blood type
system and, in some embodiments, with respect to the D blood group
system) or may be of a compatible blood group. For example, in some
embodiments type O Rh D negative blood may be administered to an
individual of any ABO blood group (i.e., A, B, O or AB), and
persons with type O RhD negative blood may be considered "universal
donors". Methods for determining blood groups are well known in the
art. In some embodiments red blood cells are cross-matched with a
potential recipient of the cells. Cross-matching may comprise
mixing a sample of the recipient's serum, plasma, or blood with a
sample of the red blood cells to potentially be administered and
checking if the mixture agglutinates. If agglutination is not
observed, the RBCs may be considered to match the recipient.
[0240] In some embodiments a eukaryotic cell that comprises a
sortagged, endogenous, non-genetically engineered polypeptide is a
genetically engineered cell. In some embodiments the cell has not
been genetically engineered for sortagging. In some embodiments the
cell has been genetically engineered before being sortagged. In
general, the cell may be any genetically engineered eukaryotic
cell. The cell may be a mammalian cell, fungal cell, insect cell,
protozoal cell, or other eukaryotic cell. The cell may be of any
cell type, e.g., any cell type described herein. For example, in
some embodiments the genetically engineered cell is an immune
system cell, e.g., a T cell, B cell, NK cell, dendritic cell,
monocyte, or macrophage. In general the cell may be genetically
engineered for any purpose, in any way, and using any method known
in the art. A genetically engineered cell may comprise multiple
genetic alterations of any one or more type(s), e.g., one or more
insertions into genomic DNA, one or more deletions of genomic DNA,
or both. A polypeptide or noncoding RNA encoded at least in part by
exogenous DNA integrated into the genome of a cell or encoded by an
endogenous gene whose sequence has been modified by genetic
engineering may be referred to as a "recombinant gene product".
[0241] In general, genetic engineering comprises introducing one or
more exogenous nucleic acids into a cell. Nucleic acids can be
introduced into cells using transfection (e.g., using any of a
variety of transfection reagents), electroporation, virus-mediated
nucleic acid transfer, etc. One of ordinary skill in the art will
select appropriate methods, vectors, expression control elements,
etc., to achieve desired alterations in the genome of a cell and/or
to achieve expression of desired proteins and/or RNAs. For example,
if a virus is used as a vector, an appropriate method may comprise
contacting a mammalian cell with the virus under conditions
appropriate for the virus to enter the cell. It will be understood
that, depending on factors such as the vector and the particular
method, a nucleic acid introduced into a cell may be at least in
part copied or reverse transcribed, and such copy or a portion
thereof may be inserted into the genome. It will also be understood
that use of the term "inserted" is not intended to imply or require
any particular mechanism and encompasses processes mediated by
retroviral integrase, homologous or non-homologous recombination,
creation of breaks in genomic DNA that are repaired by endogenous
DNA repair mechanisms, or any other process that results in
addition of one or more nucleotides to the genome of a cell or
substitution of one or more nucleotides by a different nucleotide
in the genome of a cell.
[0242] In some embodiments, a cell is genetically modified using a
nuclease that is targeted to one or more selected DNA sequences.
Such methods may be used to induce precise cleavage at selected
sites in endogenous genomic loci. Genetic engineering in which DNA
is inserted, replaced, or removed from a genome, e.g., at a defined
location of interest, using targetable nucleases, may be referred
to as "genome editing". Examples of such nucleases include
zinc-finger nucleases (ZFNs), Transcription activator-like effector
nuclease (TALENs), engineered meganuclease homing endonucleases,
and RNA directed nucleases such as CRISPR (clustered regularly
interspaced short palindromic repeats)-associated (Cas) nucleases,
e.g., derived from type II bacterial CRISPR/Cas systems (e.g.,
Cas9).
[0243] In some embodiments the nuclease comprises a DNA cleavage
domain and a DNA binding domain (DBD) that targets the nuclease to
a particular DNA sequence, thereby allowing the nuclease to be used
to engineer genomic alterations in a sequence-specific manner. The
DNA cleavage domain may create a double-stranded break (DSB) or
nick at or near the sequence to which it is targeted. ZFNs comprise
DBDs selected or designed based on DBDs of zinc finger (ZF)
proteins. DBDs of ZF proteins bind DNA in a sequence-specific
manner through one or more zinc fingers, which are regions of amino
acid sequence whose structure is stabilized through coordination of
a zinc ion. TALENs comprise DBDs selected or designed based on DBDs
of transcription activator-like (TAL) effectors (TALEs) of
Xanthomonas spp. ZFN or TALEN dimers induce targeted DNA DSBs that
stimulate DNA damage response pathways. The binding specificity of
the designed zinc-finger domain directs the ZFN to a specific
genomic site. TALEs contain multiple 33-35-amino-acid repeat
domains, each of which recognizes a single base pair. Like ZFNs,
TALENs induce targeted DSBs that activate DNA damage response
pathways and enable custom alterations. The DNA cleavage domain of
an engineered site-specific nuclease may comprise a catalytic
domain from a naturally occurring endonuclease such as the Fok1
endonuclease or a variant thereof. In some embodiments Fok1
cleavage domain variants with mutations designed to improve
cleavage specificity and/or cleavage activity may be used (see,
e.g., Guo, J., et al. (2010) Journal of Molecular Biology 400 (1):
96-107; Doyon, Y., et al., (2011) Nature Methods 8: 74-79.
Meganucleases are sequence-specific endonucleases characterized by
a large recognition site (double-stranded DNA sequences of 12 to
about 40 base pairs). The site generally occurs no more than once
in a given genome. The specificity of a meganuclease can be changed
by introducing changes sequence of the nuclease (e.g., in the DNA
binding domain) and then selecting functional enzymes capable of
cleaving variants of the natural recognition site or by associating
or fusing protein domains from different nucleases.
[0244] In some embodiments, an RNA directed nuclease may be used to
perform genome editing. For example, the use of CRISPR/Cas-based
systems is contemplated. In some embodiments a Cas nuclease, such
as Cas9 (e.g., Cas9 of Streptococcus pyogenes, Streptococcus
thermophiles, or Neisseria meningiditis, or a variant thereof), is
introduced into cells along with a guide RNA comprising a sequence
complementary to a sequence of interest (the RNA is sometimes
termed a single guide RNA). The region of complementarity may be,
e.g., about 20 nucleotides long. The Cas nuclease, e.g., Cas9, is
guided to a particular DNA sequence of interest by the guide RNA.
The guide RNA may be engineered to have complementarity to a target
sequence of interest in the genome, e.g., a sequence in any gene or
intergenic region of interest. The nuclease activity of the Cas
protein, e.g., Cas9, cleaves the DNA, which can disable the gene,
or cut it apart, allowing a different DNA sequence to be inserted.
In some embodiments multiple sgRNAs comprising sequences
complementary to different genes, e.g., 2, 3, 4, 5, or more genes,
are introduced into the same cell sequentially or together. In some
embodiments alterations in multiple genes may thereby be generated
in the same step.
[0245] In general, use of nuclease-based systems for genetic
engineering, e.g., genome editing, entails introducing a nuclease
into cells and maintaining the cells under conditions and for a
time appropriate for the nuclease to cleave the cell's DNA. In the
case of CRISP/Cas systems, a guide RNA is also introduced. The
nuclease is typically introduced into the cell by introducing a
nucleic acid encoding the nuclease. The nucleic acid may be
operably linked to a promoter capable of directing expression in
the cell and may be introduced into the cell in a plasmid or other
vector. In some embodiments mRNA encoding the nuclease may be
introduced. In some embodiments the nuclease itself may be
introduced. sgRNA may be introduced directly (by methods such as
transfection) or by expressing it from a nucleic acid construct
such as an expression vector. In some embodiments a sgRNA and Cas
protein are expressed from a single expression vector that has been
introduced into the cell or, in some embodiments, from different
expression vectors. In some embodiments multiple sgRNAs comprising
sequences complementary to different genes, e.g., 2, 3, 4, 5, or
more genes, are introduced into the same cell individually or
together as RNA or by introducing one or more nucleic acid
constructs encoding the sgRNAs into the cell for intracellular
transcription.
[0246] Upon cleavage by a nuclease, a target locus (e.g., in the
genome of a cell) may undergo one of two major pathways for DNA
damage repair, namely non-homologous end joining (NHEJ) or
homology-directed repair (HDR). In the absence of a suitable repair
template comprising sufficient homology to the sequences flanking
the cleavage site to stimulate HDR (see discussion below), DSBs are
re-ligated through NHEJ, which can result in an insertion or
deletion. NHEJ can be used, for example, to engineer gene knockouts
or generate proteins with altered activity. For example, an
insertion or deletion in an exon can lead to a frameshift mutation
or premature stop codon. Two or more DSBs can be generated in order
to produce larger deletions in the genome.
[0247] In some embodiments a nucleic acid (e.g., a plasmid or
linear DNA) comprising a sequence of interest to be inserted into
the genome at the location of cleavage is introduced into a cell in
addition to a nuclease. In some embodiments a sequence of interest
is inserted into a gene. The sequence of interest may at least in
part replace the gene. In some embodiments the nucleic acid
comprises sequences that are homologous to the sequences flanking
the cleavage site, so that homology-directed repair is stimulated.
In some embodiments the nucleic acid contains a desired alteration
as compared to a sequence present in the cell's genome at or near
the site of cleavage. A nucleic acid comprising a sequence to be at
least in part introduced into the genome, e.g., a nucleic acid
sequence comprising homologous sequence(s) and a desired alteration
may be referred to as a "donor sequence". The donor sequence may
become at least in part physically into integrated the genome at
the site of a break or may be used as a template for repair of the
break, resulting in the introduction of all or part of the
nucleotide sequence present in the donor into the genome of the
cell. Thus, a sequence in a cell's genome can be altered and, in
certain embodiments, can be converted into a sequence present in a
donor nucleic acid. In some embodiments the donor sequence may be
contained in a circular DNA (e.g. a plasmid), a linear
double-stranded DNA (e.g., a linearized plasmid or a PCR product),
or single-stranded DNA, e.g., a single-stranded oligonucleotide. In
some embodiments the donor sequence has between about 10-25 bp and
about 50-100 bp of homology to either side or each side of the
target site in the genome. In some embodiments a longer homologous
sequence may be used, e.g., between about 100-500 bp up to about
1-2 kB, or more. In some embodiments an alteration is introduced
into one allele of a gene. In some embodiments a first alteration
is introduced into one allele of a gene, and a different alteration
is introduced into the other allele. In some embodiments the same
alteration is introduced into both alleles. In some embodiments two
alleles or target sites (or more) may be genetically modified in a
single step. In some embodiments two alleles or target sites (or
more) may be genetically modified in separate steps.
[0248] Methods of designing, generating and using ZFNs and/or
TALENs are described in, e.g., WO2011097036; Urnov, F D, et al.,
Nature Reviews Genetics (2010), 11: 636-646; Miller J C, et al.,
Nat Biotechnol. (2011) 29(2):143-8; Cermak, T., et al. Nucleic
Acids Research (2011) 39 (12): e82, Sanjana, N. E. et al. A
transcription activator-like effector toolbox for genome
engineering. Nat Protoc 7, 171-192 (2012) and references in any of
the foregoing. ZFN, TALEN, and CRISPR/Cas-based methods for genome
engineering are reviewed in Gaj, T., et al., Trends Biotechnol.
2013 July; 31(7):397-405. Epub 2013 May 9. Use of CRISPR/Cas
systems in genome engineering is described in, e.g., Cong L, et al.
Multiplex genome engineering using CRISPR/Cas systems. Science.
2013; 339(6121):819-23; Mali P, et al., RNA-guided human genome
engineering via Cas9. Science. 2013; 339(6121):823-6; Wang, H. et
al. One-step generation of mice carrying mutations in multiple
genes by CRISPR/Cas-mediated genome engineering. Cell 153, 910-918
(2013); Ran, F. A. et al. Double Nicking by RNA-Guided CRISPR Cas9
for Enhanced Genome Editing Specificity. Cell 154, 1380-1389
(2013); Mali, P., et al., Nat Methods. 2013; 10(10):957-63; Ran, F
A, Nat Protoc. 2013; 8(11):2281-308). In some embodiments a
nuclease that cleaves only one strand of dsDNA (a nickase) may be
used to stimulate HDR without activating the NHEJ repair pathway.
Nickases may be created by inactivating the catalytic activity of
one nuclease monomer in the ZFN or TALEN dimer required for double
stranded cleavage or inactivating a catalytic domain of a Cas
protein. For example, mutations of one of the catalytic residues
(D10 in the RuvC nuclease domain and H840 in the HNH nuclease
domain), e.g., to alanines (D10A, H840A) convert Cas9 into DNA
nickases.
[0249] In some embodiments, a CRISP/Cas based system may be used to
modulate gene expression. For example, coexpression of a guide RNA
with a catalytically inactive Cas9 lacking endonuclease activity
generates a DNA recognition complex that can specifically interfere
with transcriptional elongation, RNA polymerase binding, or
transcription factor binding. This system, sometimes referred to
CRISPR interference (CRISPRi), can efficiently repress expression
of targeted genes in mammalian cells (Qi, S., et al., Cell. 2013;
152(5):1173-83; Larson, M H, et al., Nat Protoc. 2013;
8(10:2180-96). By attaching any of a variety of effector domains to
a catalytically inactive Cas9 one can create a chimeric Cas9
protein that can be used to achieve sequence-specific control over
gene expression and/or DNA modification. Suitable effector domains
include, e.g., a transcriptional activation domain (such as those
comprising the VP16 transactivation domain, e.g., VP64), a
transcriptional coactivation domain, a transcriptional inhibitory
or co-inhibitory domain, a protein-protein interaction domain, an
enzymatic domain, etc. A guide RNA guides the chimeric Cas9 protein
to a site of interest in the genome (e.g., in or near an expression
control element such as a promoter), whereby the effector domain
exerts an effect such as activating or inhibiting transcriptional
activity (see, e.g., Gilbert L A, et al., Cell. 2013;
154(2):442-51; Maeder M L, et al., Nat Methods. 2013;
10(10):977-9). Appropriate effector domains may be any of those
present in naturally occurring proteins that are capable of
performing the function of interest (e.g., inhibiting or activating
transcription).
[0250] Cells that have been subjected to a genetic engineering
process may be selected or analyzed to identify or isolate those
that express a desired recombinant gene product or lack expression
of an endogenous gene that has been disabled via genetic
engineering or have any desired genetic alteration. For example, in
some embodiments the donor sequence or vector used to deliver the
donor sequence may comprise a selectable marker, which may be used
to select cells that have incorporated at least a portion of the
donor sequence comprising the selectable marker into their genome.
In some embodiments selection is not used. In some embodiments
cells may be screened, e.g., by Southern blot to identify those
cells or clones that have a desired genetic alteration. If desired,
cells may be tested for expression level or activity of a
recombinant gene product or endogenous gene product or for one or
more functional properties associated with or conferred by a
recombinant or endogenous gene product, or any other criteria of
interest. Suitable methods of analysis are known to those of
ordinary skill in the art and include, e.g., Western blot, flow
cytometry, FACS, immunofluorescence microscopy, ELISA assays,
affinity-based methods in which cells are contacted with an agent
capable of binding to a protein of interest that labels or retains
cells that express the protein, etc. Functional assays may be
selected based on the identity of the recombinant gene product,
endogenous gene product, and/or function or property of interest.
For example, a functional property may be ability to bind to an
antigen of interest or ability to exert cytotoxicity towards target
cells that express an antigen of interest. Cells may be analyzed,
e.g., by PCR, Southern blotting, or sequencing, to determine the
number of inserted DNA sequences, their location, and/or to
determine whether desired genomic alterations have occurred. One or
more cells that have desired alteration(s), expression level,
and/or functional properties may be identified, propagated,
expanded. The cells or their descendants may be used to generate a
cell line, subjected to sortagging, and/or stored for future
use.
[0251] In some embodiments, a genetically engineered cell comprises
an exogenous DNA inserted into its genome. In some embodiments the
exogenous DNA comprises a sequence that encodes a polypeptide or
noncoding RNA. In some embodiments the DNA comprises a cDNA. An
inserted exogenous DNA may encode one or more RNAs or polypeptides.
In some embodiments, the genome comprises a single copy of an
exogenous DNA. In some embodiments the genome comprises no more
than 2, 3, or 4 copies. A cell may have 1, 2, 3, 4 or more distinct
DNA sequences inserted into its genome. A sequence that encodes an
RNA or polypeptide may be operably linked to appropriate expression
control elements, e.g., at least a promoter, capable of directing
transcription of the sequence. The expression control elements are
typically part of the exogenous DNA integrated into the genome.
Expression of a recombinant gene product may be constitutive or
conditional (e.g., inducible, repressible, cell-type specific). A
recombinant gene product may be any protein or RNA described
herein. In some embodiments the protein is a secreted protein, a
transmembrane protein, or an intracellular protein. In some
embodiments a cell a recombinant gene product comprises a
selectable marker e.g., an optically detectable protein such as a
fluorescent or luminescent protein or a protein that confers
resistance to a drug), a therapeutic protein, a cytokine, a
chemokine, a costimulator, a coinhibitor, a growth factor, a
receptor, or an adhesion molecule. In certain embodiments the
receptor is a chimeric antigen receptor, a T cell receptor, a B
cell receptor, a cytokine receptor, a chemokine receptor, a
costimulator receptor, an adhesion molecule, or a fusion protein
comprising two or more of the foregoing. In some embodiments a cell
is genetically engineered to produce one, more than one, or all
subunits of a multisubunit protein, e.g., a multisubunit cytokine
or receptor. In some embodiments a cell is genetically engineered
to express a noncoding RNA. A noncoding RNA may be, e.g., a short
hairpin RNA (which may be processed intracellularly to produce
siRNA), a siRNA, a microRNA precursor (which may be processed
intracellularly to produce miRNA), a miRNA, or a long noncoding
RNA. In some embodiments the noncoding RNA regulates expression of
one or more genes, e.g., by affecting transcription, processing,
stability, and/or translation of one or more pre-mRNAs or mRNAs. In
some embodiments the noncoding RNA inhibits gene expression by the
RNA interference (RNAi) pathway. By expressing an RNAi agent
targeted towards a particular gene in a cell, expression of the
gene can be stably inhibited. One of ordinary skill in the art will
be able to select appropriate sequences for an RNAi agent in order
to inhibit expression of a gene of interest.
[0252] The sequence of a recombinant gene product may be a
naturally occurring sequence or may be at least in part created by
man (non-naturally occurring) in various embodiments. In some
embodiments, the sequence comprises or consists of a full length
naturally occurring sequence (e.g., a polypeptide or RNA encoded in
a eukaryotic genome) or a functional variant or fragment thereof.
In some embodiments, the sequence comprises or consists of one or
more functional domains of a naturally occurring eukaryotic
polypeptide. In some embodiments the exogenous DNA comprises a
chimeric protein. For example, in some embodiments, the sequence
comprises or consists of multiple functional domains of different
naturally occurring eukaryotic polypeptides. In some embodiments,
the recombinant gene product comprises a sortase recognition
sequence or a sequence capable of serving as a nucleophilic
acceptor sequence in a sortase-catalyzed reaction. In some
embodiments the recombinant gene product does not comprise a
sortase recognition sequence or a sequence capable of serving as a
nucleophilic acceptor sequence in a sortase-catalyzed reaction. In
some embodiments, if a sortase recognition sequence or a sequence
capable of serving as a nucleophilic acceptor sequence in a
sortase-catalyzed reaction is present in a recombinant gene
product, it is located in an intracellular or transmembrane domain
or is otherwise not accessible to a sortase located outside the
cell.
[0253] In some embodiments exogenous DNA is integrated into the
genome of a mammalian cell, e.g., a human cell, at a "safe harbor"
locus. A "safe harbor" locus is an intragenic or extragenic region
of the genome (e.g., the human genome) that is generally able to
accommodate the insertion of DNA without causing a significant
detectable effect on the phenotype of host cell (other than the
effect, if any, caused by expression of a recombinant gene product
encoded by the inserted DNA) and that permits the transcription of
inserted DNA comprising suitable expression control elements (e.g.,
a promoter). In some embodiments, a significant effect is a
statistically significant change in the viability or proliferative
capacity of the cell or the ability of the cell to perform a normal
biological function. In some embodiments a safe harbor locus is the
AAVSV1 (the natural integration site for the wild-type AAV on
chromosome 19), ROSA26, or CCR5 locus. The locations of these loci
are well known in the art. The AAVS1 site is in chromosome 19
(position 19q13.42) and integration in the AAVS1 locus may disrupt
the gene phosphatase 1 regulatory subunit 12C (PPP1R12C). The human
ROSA26 locus is in chromosome 3 (position 3p25.3). The human CCR5
gene is located on chromosome 3 (position 3p21.31). See US Pat.
Pub. 20110239319 for a description of additional sites that may be
used as safe harbor loci, methods of identifying safe harbor loci,
and methods of inserting DNA into safe harbor loci. In some
embodiments exogenous DNA is integrated into a site located at
least 100 kB away from any known proto-oncogene or tumor suppressor
gene.
[0254] In some embodiments a cell is genetically engineered to
alter the expression or sequence of an endogenous gene. For
example, in some embodiments a gene whose expression is not desired
is disabled in the cell. As used herein, a gene is "disabled" in a
cell if its expression is reduced to a level below that which is
necessary for the cell to exhibit normal levels of one or more
biological activities of the gene product or if the sequence of the
encoded gene product is altered in a way that reduces the activity
of the gene product to a level below that which is necessary for
the cell to exhibit normal levels of one or more biological
activities of the gene product. A gene may be disabled by at least
partly deleting the gene, by introducing an insertion into the gene
in an appropriate location, or by altering the gene so that the
encoded gene product has reduced activity, e.g., by deleting or
changing an active site residue or other functionally important
residue. In some embodiments, precise alterations that introduce
one or more substitutions, insertions, or deletions at a desired
location are made. Such alterations may, for example change the
sequence of an allele that causes or contributes to a disease to
one that is not associated with the disease or may change the
specificity of a receptor. Expression control elements may be
inserted or modified to increase or decrease expression of a
selected endogenous gene. In some embodiments the endogenous gene
encodes a receptor, a transmembrane protein, a secreted protein, a
costimulator, a coinhibitor, a cytokine, a chemokine, a growth
factor, or an adhesion molecule. In some embodiments an endogenous
gene that is disabled may encode a gene product that mediates
immunosuppressive extracellular signals (e.g., receptors for
cytokines that may exert immunosuppressive effects, such as IL-10
or TGF-beta) or contributes to T cell exhaustion. T cell exhaustion
is a state of T cell dysfunction that arises during many chronic
infections and cancer. It is defined by poor effector function,
sustained expression of inhibitory receptors and a transcriptional
state distinct from that of functional effector or memory T cells
(reviewed in Wherry, E J, Nature Immunology, 2011; 12(6):492-9). T
cell exhaustion can be promoted by a variety of cell surface
inhibitory receptors such as PD-1, LNG-3, CD244 (2B4), CD160,
TIM-3, and/or CTLA-4. In some embodiments inhibiting one or more
such receptors, e.g., by disabling one or more genes that encode
such receptors, may reduce the likelihood that a T cell will become
exhausted. In some embodiments an endogenous gene that is disabled
may encode a gene product that mediates effects of a toxic
substance such as a pro-apoptotic agent, a cytolytic agent, a
cytotoxic drug, or a toxin. For example, the endogenous gene may
encode a receptor for the toxic substance. Disabling the endogenous
gene may reduce the susceptibility of the cell to the substance
relative to a cell in which the endogenous gene is expressed or
functional.
[0255] As discussed herein, certain embodiments relate to sortagged
mammalian immune system cells that are capable of mounting an
immune response towards target cells that are recognized by the
sortagged immune system cells. For example, certain embodiments
relate to sortagged mammalian immune system cells that comprise a
binding moiety that binds to an antigen expressed by target cells.
In some embodiments, such sortagged immune system cells have
cytotoxic activity towards target cells. The cells may, for
example, be CD8+ T cells or NK cells. In some embodiments such
cells are not genetically engineered.
[0256] In some embodiments immune system cells are genetically
modified to express a recombinant gene product that binds to an
antigen of interest, e.g., an antigen expressed by target cells.
For example, in some embodiments a recombinant gene product
comprises a chimeric antigen receptor that binds to a particular
antigen of interest. The cells may, in addition, be sortagged with
any of the various agents described herein. In some embodiments a
recombinant gene product comprises a TCR chain that hinds to a
particular antigen of interest. The TCR chain(s) may originate from
a T cell with high affinity for a selected antigen. The T cell may
originate from a human or from a non-human mammal following
immunization with the antigen or a portion thereof). The non-human
mammal may be genetically engineered to express human TCR genes
(e.g., at least a TCR alpha and/or beta chain). T cells that bind
to an antigen of interest may be identified, and their TCR genes
may be isolated and, optionally, may be sequenced at least in part.
Human TCR genes with a desired affinity to a selected antigen may
alternately or additionally be identified using display
technologies such as phage display. In some embodiments, an
endogenous gene that encodes a TCR chain may be modified by
introducing one or more alterations that cause the binding
specificity of the endogenous TCR to be directed to an antigen of
interest. For example, at least a portion of the variable domain of
an endogenous TCR gene may be modified. In some embodiments one or
more CDRs may be modified. Introducing genes encoding TCRalpha and
TCRbeta chains that have affinity for a selected target antigen
into the genome of a. T cell or modifying endogenous TCR chains to
confer or increase affinity for a selected target antigen creates a
redirected T cell that is capable of recognizing and responding to
the target antigen. In some embodiments one or more chains of an
endogenous TCR gene may be disabled. Disabling an endogenous TCR
may be useful, e.g., when a cell is engineered to express a CAR or
when a gene encoding an exogenous TCR chain is introduced into a
site other than the site of the corresponding endogenous TCR chain.
Similar methods may be applied to TCR delta and gamma chains and/or
BCR chains. Certain methods and compositions useful for introducing
desired TCR genes into a known chromosomal locus a safe harbor
locus), altering endogenous TCR genes, and/or inactivating TCR
genes using zinc finger nucleases (ZFNs) are described in U.S.
Publication No. 20110158957.
[0257] A recombinant gene product may serve any one or more
purposes described herein. For example, a recombinant gene product
may serve as a binding moiety, targeting moiety, therapeutic agent,
detectable agent, or agent that alters one or more properties of
the cell. In some embodiments, an agent conjugated to the cell
using sortase comprises a targeting moiety, and the recombinant
gene product comprises a chimeric antigen receptor. In some
embodiments, an agent conjugated to the cell using sortase
comprises a targeting moiety, and the recombinant gene product
comprises a protein or RNA that alters one or more properties of
the cell. Altering one or more properties of the cell may comprise
stimulating or inhibiting proliferation or activity of the cell,
inhibiting or enhancing activity of a cell surface receptor,
conferring responsiveness to an extracellular ligand. The
recombinant gene product may increase or inhibit expression of one
or more genes, bind to an intracellular domain of a receptor,
increase or inhibit an intracellular protein-protein interaction,
increase or inhibit an intracellular protein translocation. In some
embodiments, an agent conjugated to the cell using sortase
comprises a targeting moiety, and the recombinant gene product
comprises a different targeting moiety. The first and second
targeting moieties may be the same or different. If different, they
may bind to the same antigen (e.g., to different epitopes thereof)
or to different antigens. For example, the first and second
targeting agents may bind to different tumor antigens, which may
both be present in a tumor. In some embodiments, an agent
conjugated to the cell using sortase comprises a targeting moiety,
and the recombinant gene product comprises a therapeutic protein.
In some embodiments the targeting moiety targets the cell to a
desired site of activity in the body.
[0258] In some embodiments, immune system cells that are to be
administered to a subject or contacted with other cells ex vivo may
be processed to reduce the likelihood that they will mount an
immune response against non-target cells and/or to reduce the
likelihood that a subject's immune system cells will mount an
immune response towards administered cells. In some embodiments, T
cells are rendered nonreactive to antigens that are present on
normal cells of a subject (alloantigens) by coculturing them with
allogeneic cells in the presence of one or more agents that inhibit
costimulation of the T cells. The nonreactive cells may be referred
to as "anergized cells". The allogeneic cells may be obtained from
a subject to whom the anergized cells are to be administered. In
some embodiments the allogeneic cells are allogeneic PBMC.
Costimulation can be inhibited by, e.g., inhibiting the receipt of
costimulatory signals. This may be achieved by blocking the binding
of B7 family members (e.g., B7-1 and/or B7-2) to their receptors on
T cells using agents such as anti-B7-1 and/or anti-B7-2 monoclonal
antibodies. The anergized cells preferably retain ability to
recognize and kill target cells but have reduced or absent ability
to kill non-target cells. In some embodiments.
[0259] In some embodiments, expression or activity of an endogenous
TCR is eliminated or inhibited, which may be achieved, for example,
by engineering a disruption or insertion in a gene encoding TCR
.alpha., TCR .beta., CD3zeta, and/or CD3epsilon. In some
embodiments, the cell may also be genetically engineered to express
a TCR or a CAR. In some embodiments, immune system cells in which
endogenous TRC expression or activity is eliminated or inhibited
lack ability to respond to TCR-mediated stimulation but retain
ability to be stimulated by contact with an antigen to which a TCR
or CAR has specificity. (See Torikai, H., et al., Blood (2012),
119(24): 5697-5705 for description of irreversible disruption of
endogenous TCR expression in CAR T cells using zinc finger
nucleases targeting the constant regions of TCR .alpha. or TCR
.beta. genes. The CAR T cells expressed a CAR comprising a moiety
that binds to CD19. The TCRnegCAR+ T cells did not respond to
TCR-mediated stimulation by cross-linking CD3 with OKT3, but
retained CD19 specificity, were activated and stimulated to
proliferate by contact with CD19, and induced cytotoxicity in CD19+
leukemic cells.) Such an approach may be used, e.g., to generate
allogeneic antigen-specific T cells from one donor that may be
administered to multiple different recipients. In some embodiments
cells are engineered to express one or more nucleic acids encoding
shRNA, siRNA, and/or miRNA molecules to down-regulate expression of
an endogenous gene encoding TCR .alpha., TCR .beta., CD3zeta,
and/or CD3epsilon. In some embodiments, immune system cells in
which endogenous TRC expression or activity is eliminated or
inhibited may be administered to subjects who express different
major and/or minor histocompatibility antigens than do the
administered cells, with reduced likelihood of resulting in
graft-versus-host-disease (GVHD) as compared with administration of
control cells that express a functional endogenous TCR.
[0260] In some embodiments cells, e.g., cells to be administered to
a subject, are engineered to eliminate or inhibit expression of one
or more HLA class I and/or II genes, e.g., genes encoding HLA-A,
HLA-B, HLA-C, or encoding an alpha or beta chain of HLA-DR, HLA-DP,
or HLA-DQ. In some embodiments, this is achieved by engineering a
disruption or insertion in the gene or by engineering cells to
express one or more nucleic acids encoding shRNA, siRNA, and/or
miRNA molecules that inhibit expression of the gene. In some
embodiments, eliminating or inhibiting expression or activity of
HLA class I and/or II genes of cells to be administered to a
subject may reduce the likelihood that endogenous immune system
cells of a subject will mount an immune response against the
administered cells.
[0261] In some embodiments, cells that are engineered to lack
expression of one or more endogenous cell-surface proteins may be
contacted with one more agents (e.g., antibodies) that binds to the
extracellular portion of such proteins in order to remove remaining
cells (if any) that express the protein at their surface. The
binding agent may be attached to a support, such as magnetic beads,
which retains any cells that express the endogenous protein at
their surface For example, cells engineered to lack expression of
the TCR may be contacted with anti-CD3 antibodies to remove
remaining cells that express a TCR at their cell surface.
[0262] In some embodiments a cell is genetically engineered to
comprise a gene that encodes an inducible suicide gene. A suicide
gene is a gene that encodes a gene product that causes a cell that
contains the suicide gene gene to die following induction of
expression of the gene or induction of activity of the gene product
encoded by the gene. In certain embodiments at least 50%, 60%, 70%,
80%, 90%, 95%, 97%, 98%, 99%, or more of the cells have died within
a defined time period following induction of expression or activity
of the gene or induction of activity of the gene product, The
defined time period may be, e.g., about 24-72 hours. In some
embodiments, the inducible suicide gene is expressed only in the
presence of an inducer, which may be contacted with the cells ex
vivo or administered to a subject to whom cells comprising the
suicide gene have been administered. In some embodiments, the
inducible suicide gene may be expressed in the absence of an
inducer, but the resulting gene product is inactive in the absence
of the inducer. Administration of the inducer to a subject
comprising cells that harbor an inducible suicide gene causes
induction of the suicide gene, resulting in death or at least
reduced proliferation of cells that harbor the suicide gene. In
some embodiments the protein encoded by the suicide gene has one,
more than one, or all of the following properties: non-immunogenic
(at least in humans or other mammalian subjects to whom a cell
expressing the suicide gene may be administered), non-cell-cycle
dependent (the transcription of the gene and activity of the gene
product are not restricted to particular phases of the cell cycle
or limited to dividing cells or inactive in quiescent cells),
clinically compatible (neither the gene product nor the inducing
agent produce unacceptable toxicity in vivo), inducible by an
inducing agent that has wide biodistribution so that the inducing
agent, when administered to a subject, will reach a high percentage
of cells harboring the gene. In some embodiments, a gene that
encodes at least a portion of a human protein may be used. In some
embodiments an inducible suicide gene exploits chemical inducers of
dimerization (CID) (see, e.g., Amara J F, Proc Natl Acad Sci USA.
1997; 94(20):10618-23; Clackson T, et al., Proc Natl Acad Sci USA.
1998, 95:10437-10442; Rollins C T, et al. Proc Natl Acad Sci USA.
2000; 97(13):7096-7101). According to this approach, a proapototic
molecule or other inducer of cell death is modified to comprise one
or more binding sites for a CID. Binding of the CID to its
target(s) causes their oligomerization, resulting in activation of
the apoptotic pathway or other cell death-inducing pathway. In some
embodiments the binding site for a CID comprises a mutated
FK506-binding protein (FKBP12) that mediates dimerization upon
binding of a small molecule ligand (e.g., a CID such as AP1510 or
AP1903). In some embodiments the CID is a dimeric analog of the
immunosuppressive agent FK506. In some embodiments the analog of
FK506 lacks the immunosuppressive activity of FK506. In some
embodiments the CID is biologically inert at concentrations at
which it is used. Fusing one or more FKBPs, e.g., FKBP12 or a
mutant thereof to a pro-apoptotic domain or other cell death
inducing domain, results in a protein whose activity (pro-apoptotic
activity or other cell death inducing activity) can be stimulated
by a CID. If the protein is expressed by a cell, activity of the
protein can be stimulated by contacting the cell with a CID, e.g.,
by culturing the cell in the presence of a CID or administering the
CID to a subject comprising the cell. Examples of useful suicide
genes include genes that encode pro-apoptotic proteins, which can
be modified to render their activity inducible. For example,
inducible suicide genes based on caspases 1, 3, 8, or 9 (Straathof
K C, et al. Blood. 2005; 105(10:4247-4254), the death receptor Fas
(Thomis D C, et al., Blood. 2001; 97(5):1249-1257) or FADD may be
used. One such gene, designated iC9, encodes a fusion protein that
links a truncated human caspase 9 lacking the endogenous caspase
recruitment domain (CARD) with a mutated FK506-binding protein
(FKBP12). In the presence of an appropriate CID, e.g., AP1903,
functional active caspase 9 is generated, leading to apoptosis
(see, e.g., Fan L, et al., (1999) Hum Gene Ther 10: 2273-2285; see
also 20110286980). In general, an inducible suicide gene may be
introduced into cells using standard methods of genetic
engineering. For example, a nucleic acid encoding the gene product
operably linked to a promoter may be introduced in a vector, e.g.,
a viral vector or plasmid, which is then introduced into a cell. In
the case of a cell that is genetically engineered to express a CAR,
the nucleic acid encoding the inducible suicide gene product may in
some embodiments be included in the same construct or vector,
optionally further including a sequence encoding a cytokine such as
IL-15. Examples of cells that genetically engineered to express a
CAR, an inducible suicide gene, and a cytokine are described in
U.S. Pat. Pub. No. 20130071414.
[0263] In some embodiments, two or more nucleic acid sequences that
encode different proteins, different noncoding RNAs, or at least
one protein and at least one noncoding RNA, are included in a
single nucleic acid construct, which may further include one or
more operably linked expression control elements. Without wishing
to be bound by any theory, may be advantageous as it allows
coexpression of multiple gene products from a single exogenous DNA.
In some embodiments, transcription of each nucleic acid sequence
may be directed by a promoter operably linked thereto, resulting in
two or more separate RNAs. In some embodiments, a bidirectional
promoter may be used to direct transcription of two separate RNAs.
In some embodiments, a single promoter directs expression of an RNA
that encodes multiple (e.g., two, three, four, or more)
polypeptides. Translation of multiple polypeptides from one RNA can
be achieved by using self-cleaving peptides or internal ribosome
entry sites (IRESs). The sequences that encode the polypeptides may
have an internal ribosome entry site (IRES) located between them or
a sequence that encodes a "self-cleaving peptide" such as a 2A
peptide. When IRES elements are included between multiple open
reading frames (ORFs), the first ORF is translated by the typical
cap-dependent mechanism, while the rest are translated through a
cap-independent mechanism (Martinez-Salas, E. Curr Opin Biotechnol.
1999; 10: 458-464; Hellen C U, Sarnow P, Genes & Development,
2001; 15: 1593-1612). Since the genes are translated independently,
the relative expression of different genes can be adjusted, if
desired, by varying the strength of the IRES located upstream of
each ORF. IRESs are found in a variety of different viral (e.g.,
picornavirus) and eukaryotic mRNAs. For example, IRESes are found
in entero- and rhinoviruses, cardio- and aphthoviruses, and
hepatitis A virus. The encephalomyocarditis virus (EMCV) IRES is
among the most widely used IRES elements for multiple gene
expression in mammalian cells. An exemplary IRES sequence comprises
about the region from 260 to 848 in the EMCV-R genome (Genbank:
M81861). Other IRES sequences useful for expressing multiple open
reading frames encoding different polypeptides from a single
promoter are described in Sasaki Y, J Biotechnol. 2008;
136(3-4):103-12. IRES sequences may be identical to those found in
nature or may be modified to increase or decrease their efficiency
and thereby alter the absolute and/or relative amount of the linked
ORFs. Self-cleaving 2A peptides mediate `ribosomal skipping`
between the proline and glycine residues and inhibit peptide bond
formation without affecting downstream translation. These peptides
allow multiple proteins to be encoded as polyproteins, which
dissociate into component proteins upon translation. Sequences
linked by 2A peptides are expressed in a single open reading frame
(ORF) and "self-cleavage" occurs co-translationally to produce
separate polypeptides. Use of the term "self-cleaving" in reference
to 2A peptides is common in the art and is not intended to imply a
proteolytic cleavage reaction. Multicistronic vectors comprising 2A
peptides between sequences that encode proteins are reviewed in
Szymczak A L, et al. 2005; 5:627-638). Self-cleaving peptides are
found in members of the Picornaviridae virus family, including
aphthoviruses such as foot-and-mouth disease virus (FMDV), equine
rhinitis A virus (ERAV), Thosea asigna virus (TaV) and porcine
teschovirus-1 (PTV-1) and cardioviruses such as Theilovirus (e.g.,
Theiler's murine encephalomyelitis) and encephalomyocarditis
viruses (Donnelly, M L, et al., J. Gen. Virol., 2001; 82:
1027-1041; Ryan, M D, et al., J. Gen. Virol., 2001; 72: 2727-2732;
DeFelipe, P., et al., Trends Biotechnol. 2006; 24(2):68-75). The 2A
peptides derived from FMDV, ERAV, PTV-1, and TaV are sometimes
referred to as "F2A", "E2A", "P2A", and "T2A", respectively.
Aphthovirus 2A polypeptides contain a Dx1Ex2NPG sequence (SEQ ID
NO: 5), where x1 is often valine or isoleucine. An exemplary 2A
sequence is VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 6) from FMDV, where
underlined residues are conserved in many 2A peptides. In some
embodiments a spacer sequence such as GSG or SGSG may be included
ahead of a 2A sequence. In some embodiments a protease cleavage
site may additionally be included, such as a cleavage site for
furin (RAKR). A cell may be genetically engineered to express any
two or more recombinant gene products using any of the above
approaches. In some embodiments two or more subunits of a
multisubunit protein are expressed. In some embodiments, any two or
more of the following recombinant gene products are expressed: a
receptor, a cytokine, a costimulator, a coinhibitor, a suicide
protein. The receptor may be an antigen receptor (e.g., a chimeric
antigen receptor), a cytokine receptor, a costimulatory or
coinhibitory receptor. All different combinations of products to be
expressed and methods of achieving expression of multiple products
are encompassed. In some embodiments, for example, a CAR and a
suicide protein, a CAR and a cytokine, or a CAR, a cytokine, and a
suicide protein may be translated from a single RNA encoded by an
exogenous DNA inserted into the genome of a cell.
V. Click Chemistry
[0264] In some embodiments an agent conjugated or to be conjugated
to a eukaryotic cell, e.g., a mammalian cell, may comprise a click
chemistry handle. For example, A.sup.1 in a sortase substrate
described above may comprise a click chemistry handle. Click
chemistry is a chemical philosophy introduced by Sharpless in 2001
and describes chemistry tailored to generate substances quickly and
reliably by joining small units together (see, e.g., Kolb, Finn and
Sharpless Angewandte Chemie International Edition (2001) 40:
2004-2021; Evans, Australian Journal of Chemistry (2007) 60:
384-395). Additional exemplary click chemistry handles, reaction
conditions, and associated methods useful according to aspects of
this invention are described in Joerg Lahann, Click Chemistry for
Biotechnology and Materials Science, 2009, John Wiley & Sons
Ltd, ISBN 978-0-470-69970-6. In some embodiments, a click chemistry
handle is described in any of the references herein and/or in Table
1 or Table 2. For example, a click chemistry handle may comprise or
consist of a terminal alkyne, azide, strained alkyne, diene,
dieneophile, alkoxyamine, carbonyl, phosphine, hydrazide, thiol, or
alkene moiety.
[0265] Two entities, e.g., two proteins, each comprising a click
chemistry handle (e.g., a first protein comprising a click
chemistry handle providing a nucleophilic (Nu) group and a second
protein comprising an electrophilic (E) group that can react with
the Nu group of the first click chemistry handle) can be covalently
conjugated under click chemistry reaction conditions. The
installation of a click chemistry handle on a protein confers click
chemistry reactivity to the protein. In some embodiments a
sortase-mediated conjugation is used to install a first click
chemistry handle on a polypeptide expressed by a mammalian cell,
and a click chemistry reaction is then used to conjugate an entity
comprising a second click chemistry handle to the first click
chemistry handle, thereby conjugating the entity to the polypeptide
and thus attaching it to the cell. In general, the second click
chemistry handle may be located at any position of the entity. In
some embodiments the entity comprising the second click chemistry
handle is a polypeptide. In some embodiments the second click
chemistry handle may be at the C-terminus or the N-terminus of the
polypeptide or may be attached to a side chain at or near the
C-terminus or N-terminus. The second click chemistry handle may be
incorporated into the entity, e.g., polypeptide using sortase or
other methods. Methods of installing click chemistry handles on
polypeptides are described in PCT/US2012/044584. In some
embodiments the use of click chemistry allows two proteins to be
conjugated at their respective N-termini, generating an N--N
conjugated chimeric protein. For example, the N-terminus of a
non-genetically engineered polypeptide expressed by a mammalian
cell may be modified using sortase to install a first click
chemistry handle, as described herein. A polypeptide having a
compatible click chemistry handle installed at or near its
N-terminus may then be conjugated via click chemistry to the first
click chemistry handle.
[0266] Click chemistry should be modular, wide in scope, give high
chemical yields, generate inoffensive byproducts, be
stereospecific, be physiologically stable, exhibit a large
thermodynamic driving force (e.g., >84 kJ/mol to favor a
reaction with a single reaction product), and/or have high atom
economy. Several reactions have been identified which fit this
concept:
[0267] (1) The Huisgen 1,3-dipolar cycloaddition (e.g., the
Cu(I)-catalyzed stepwise variant, often referred to simply as the
"click reaction"; see, e.g., Tornoe et al., Journal of Organic
Chemistry (2002) 67: 3057-3064). Copper and ruthenium are the
commonly used catalysts in the reaction. The use of copper as a
catalyst results in the formation of 1,4-regioisomer whereas
ruthenium results in formation of the 1,5-regioisomer;
[0268] (2) Other cycloaddition reactions, such as the Diels-Alder
reaction;
[0269] (3) Nucleophilic addition to small strained rings like
epoxides and aziridines;
[0270] (4) Nucleophilic addition to activated carbonyl groups;
and
[0271] (5) Addition reactions to carbon-carbon double or triple
bonds.
[0272] For two proteins to be conjugated via click chemistry, the
click chemistry handles of the proteins have to be reactive with
each other, for example, in that the reactive moiety of one of the
click chemistry handles can react with the reactive moiety of the
second click chemistry handle to form a covalent bond. Such
reactive pairs of click chemistry handles are well known to those
of skill in the art and include, but are not limited to those
described in Table I:
TABLE-US-00006 TABLE 1 Exemplary click chemistry handles and
reactions. ##STR00010## ##STR00011## ##STR00012## ##STR00013##
##STR00014##
[0273] In some embodiments R, R.sub.1 or R.sub.2 in click chemistry
handles and reactions above is a non-genetically engineered
polypeptide -[Xaa].sub.y-TRS-PRT according to Formula I above
expressed by a living mammalian cell, wherein the polypeptide has
been modified by sortase-catalyzed conjugation of a click chemistry
handle thereto, and the other of R, R.sub.1 or R.sub.2 is a moiety
to be conjugated to the click chemistry handle of the modified
polypeptide.
[0274] In some embodiments, click chemistry handles are used that
can react to form covalent bonds in the absence of a metal
catalyst. Such click chemistry handles are well known to those of
skill in the art and include the click chemistry handles described
in Becer, Hoogenboom, and Schubert, Click Chemistry beyond
Metal-Catalyzed Cycloaddition, Angewandte Chemie International
Edition (2009) 48: 4900-4908.
TABLE-US-00007 TABLE 2 exemplary click chemistry handles and
reactions. Reagent A Reagent B Mechanism Notes on reaction.sup.[a]
Reference 0 azide alkyne Cu-catalyzed [3 + 2] 2 h at 60.degree. C.
in H.sub.2O [9] azide-alkyne cycloaddition (CuAAC) 1 azide
cyclooctyne strain-promoted [3 + 2] 1 h at RT [6-8, azide-alkyne
cycloaddition 10, 11] (SPAAC) 2 azide activated [3 + 2] Huisgen 4 h
at 50.degree. C. [12] alkyne cycloaddition 3 azide electron- [3 +
2] cycloaddition 12 h at RT in H.sub.2O [13] deficient alkyne 4
azide aryne [3 + 2] cycloaddition 4 h at RT in THF [14, 15] with
crown ether or 24 h at RT in CH.sub.3CN 5 tetrazine alkene
Diels-Alder retro- 40 min at 25.degree. C. [36-38] [4 + 2]
cycloaddition (100% yield) N.sub.2 is the only by-product 6
tetrazole alkene 1,3-dipolar cycloaddition few min UV radiation and
[39, 40] (photoclick) then overnight at 4.degree. C. 7 dithioester
diene hetero-Diels-Alder 10 min at RT [43] cycloaddition 8
anthracene maleimide [4 + 2] Diels-Alder 2 days at reflux in
toluene [41] reaction 9 thiol alkene radical addition 30 min UV
(quantitative conv.) [19-23] (thio click) or 24 h UV irradiation
(>96%) 10 thiol enone Michael addition 24 h at RT in CH.sub.3CN
[27] 11 thiol maleimide Michael addition 1 h at 40.degree. C. in
THF or [24-26] 16 h at RT in dioxane 12 thiol para-fluoro
nucleophilic substitution overnight at RT in DMF or [32] 60 min at
40.degree. C. in DMF 13 amine para-fluoro nucleophilic substitution
20 min MW at 95.degree. C. in [30] NMP as solvent .sup.[a]RT = room
temperature, DMF = N,N-dimethylformamide, NMP = N-methylpyrolidone,
THF = tetrahydrofuran, CH.sub.3CN = acetonitrile.
From Becer, Hoogenboom, and Schubert, click Chemistry beyond
Metal-Catalyzed Cycloaddition, Angewandte Chemie International
Edition (2009) 48: 4900-4908.
[0275] Additional click chemistry handles suitable for use in
methods of conjugation described herein are well known to those of
skill in the art, and such click chemistry handles include, but are
not limited to, the click chemistry reaction partners, groups, and
handles described in PCT/US2012/044584 and references therein,
which references are incorporated herein by reference for click
chemistry handles and methodology.
[0276] In some embodiments eukaryotic cells, e.g., mammalian cells
are modified by using sortase to conjugate a moiety comprising a
first click chemistry handle to a polypeptide expressed by the
cells, wherein the polypeptide is not genetically engineered to
comprise an extracellular sortase recognition motif or
extracellular glycine, e.g., non-genetically engineered eukaryotic,
e.g., mammalian, cells. Once a click chemistry handle has been
installed using sortase, the cells may be further modified by click
chemistry mediated attachment of any available entity comprising a
compatible click chemistry handle, without need to modify the
entity to incorporate a sortase recognition motif. This approach
may facilitate rapid conjugation of diverse entities onto cells. In
some embodiments a population of cells that have been modified by
using sortase to conjugate a moiety comprising a first click
chemistry handle to a polypeptide expressed by the cells may be
divided into two or more aliquots. The number of aliquots and
number of cells per aliquot may be selected in any convenient
manner. In some embodiments aliquots comprise at least 10.sup.3,
10.sup.4, 10.sup.5, 10.sup.6, or 10.sup.7 cells. In some
embodiments the number of aliquots is between 2 and 1,000. One or
more aliquots may be stored for future use. In some embodiments two
or more different moieties, each comprising a second click
chemistry handle that is compatible with the first click chemistry
handle are conjugated to cells of two or more different aliquots,
to produce two or more populations of modified mammalian cells
having the different moieties conjugated thereto. Cells of
different aliquots, or portions thereof, having different entities
conjugated thereto, may be subsequently combined.
VI. Sortagged Mammalian Cells and Uses Thereof
[0277] Without limiting the invention, this section discusses
certain proteins, nucleic acids, lipids, small molecules, and other
entities of interest in the context of the present disclosure,
certain methods of preparing or using sortagged mammalian cells,
and related compositions. Entitites described herein may be used
for various purposes. For example, in various embodiments proteins,
nucleic acids, lipids, small molecules, and sugars may be
conjugated to mammalian cells using sortase, conjugated to each
other using sortase, used in cell culture (e.g., to expand,
stimulate, or differentiate cells), and/or administered to a
subject.
[0278] In some embodiments a first protein is conjugated to a
second protein using sortase. In some embodiments the second
protein is expressed by a living mammalian cell. For example,
A.sup.1 in sortase substrate A described above may comprise any
protein. In general, any protein or polypeptide that comprises or
is modified to comprise an appropriately positioned sortase
recognition motif can be conjugated to a living mammalian cell. In
some embodiments a protein is modified by conjugating an agent to
it using sortase, and the resulting protein is conjugated to a
mammalian cell using sortase. In some embodiments two or more
polypeptides are conjugated using sortase, and the resulting
protein is conjugated to a mammalian cell using sortase. In some
embodiments a polypeptide may be extended to include a sortase
recognition motif at or near its C-terminus and/or to include one
or more N-terminal glycines or other appropriate nucleophilic
acceptor sequence to allow it to participate in a sortase-catalyzed
reaction.
[0279] In some embodiments a protein is an enzyme, e.g., an enzyme
that plays a role in metabolism or other physiological processes in
a mammal. In some embodiments a protein, is characterized in that
deficiency of the protein underlies a disease that affects a
mammal. In some embodiments a protein is an enzyme that plays a
role in carbohydrate metabolism, amino acid metabolism, organic
acid metabolism, porphyrin metabolism, purine or pyrimidine
metabolism, and/or lysosomal storage. Deficiencies of enzymes or
other proteins can lead to a variety of diseases, e.g., diseases
associated with defects in carbohydrate metabolism, amino acid
metabolism, organic acid metabolism, purine or pyrimidine
metabolism, lysosomal storage disorders, and blood clotting, among
others. Examples include the following (name of deficient enzyme or
other protein is indicated in parentheses if not part of name of
disease): glucose-6-phosphate dehydrogenase deficiency, alpha-1
antitrypsin deficiency, phenylketonuria (deficiency in
phenylalanine hydroxylase), Fabry disease (alpha galactosidase A
deficiency), Gaucher disease (glucocerebrosidase deficiency), Pompe
disease (acid alpha-glucosidase deficiency), adenosine deaminase
deficiency, mucopolysaccharidoses such as MPSI (alpha-L-iduronidase
deficiency), MPSII (iduronate-2-sulfatase deficiency), MPSIIIA
(heparan sulfamidase deficiency), MPSVI
(N-acetylgalactosamine-4-sulfatase deficiency), hemophilia (various
coagulation factors), hereditary angioedema (C1 esterase inhibitor
deficiency); hypophosphatasia (tissue-nonspecific isozyme of
alkaline phosphatase (TNSALP). In some embodiments the enzyme is
one wherein exogenous administration of the enzyme at least in part
alleviates the disease. In some embodiments the enzyme is one that
is normally present in the blood. In some embodiments the enzyme
may normally be produced by cells in the liver or kidneys and
secreted into the blood. In some embodiments the enzyme acts on a
substrate whose increased presence or accumulation in the blood may
contribute to a disease or which may be transported by the blood to
a site in the body where it contributes to a diseases. In some
embodiments a deficiency is due to an inherited mutation. In some
embodiments a deficiency may be transient, e.g., it may be due at
least in part to excessive consumption, degradation, or loss of the
enzyme (e.g., due to bleeding), or due at least in part to toxicity
(e.g., exposure to a toxin that inactivates the enzyme or increases
requirement for the enzyme). In some embodiments a disease, e.g.,
an enzyme deficiency disease, is a rare disease or orphan disease
as defined in the US or as defined in the relevant jurisdiction
where a patient is treated.
[0280] In some embodiments an enzyme conjugated to mammalian cells
is catalytically active. In some embodiments the enzyme is in a
catalytically inactive form (e.g., a zymogen) and is cleaved in
vivo (after administration of the cells to a subject) to generate
an active form. Such cleavage may be catalyzed by an endogenous
protease. In some embodiments the enzyme may be released from cells
in vivo. Such release may occur via a proteolytic cleavage that, in
some embodiments, also activates the enzyme. It will be understood
that an enzyme used to treat an enzyme deficiency need not be the
same as the deficient enzyme so long as it provides the required
enzymatic activity, e.g., at least the enzymatic activity necessary
to treat the disease.
[0281] In some embodiments an agent comprises both (i) a
therapeutically active domain, e.g., an enzyme, small molecule,
therapeutic protein, therapeutic antibody, and (ii) a targeting
domain, wherein the targeting domain targets the cells and/or agent
to a site in the body where the therapeutic activity is desired.
The targeting domain binds to a target present at such site. Any
targeting domain may be used, e.g., an antibody. In some
embodiments the agent may be released from cell surfaces after
administration and the released agent, comprising both a targeting
domain and a therapeutic domain, accumulates at a site of disease.
The site may be any organ or tissue, e.g., any organ or tissue
where the disease causes destruction, degradation, or symptoms. For
example, an agent may be targeted to respiratory tract (e.g.,
lung), bone, kidney, liver, pancreas, skin, cardiovascular system
(e.g., heart), smooth or skeletal muscle, gastrointestinal tract,
eye, blood vessel surfaces, etc.
[0282] In some embodiments an agent conjugated to mammalian cells,
e.g., hematopoietic cells, e.g., red blood cells, comprises a
moiety that modulates blood coagulation or breakdown of blood clots
(fibrinolysis). In some embodiments the moiety promotes blood
coagulation, e.g., the moiety may be a protein that participates in
the coagulation pathway, e.g., a coagulation factor (e.g., factor
VII, VIIa, VIII or IX). In some embodiments, mammalian cells
conjugated with a moiety that promotes blood coagulation, such as a
coagulation factor, may be administered to a subject to speed blood
clotting in order to promote cessation of blood loss from a damaged
vessel (e.g., a subject who has experienced a physical injury). In
some embodiments cells conjugated with an agent that promotes blood
coagulation may be administered prophylactically, e.g., to a
subject with a defect in blood coagulation (e.g., hemophilia) with
an aim of preventing excessive blood loss in case the subject is
injured, to a subject who is to undergo surgery that presents a
risk of significant blood loss, or to a subject who has received an
excessive amount of an anticoagulant. In some embodiments the
moiety inhibits coagulation and/or promotes fibrinolysis, e.g., the
moiety may comprise a coagulation pathway regulator, heparin,
plasmin, tissue plasminogen activator, streptokinase, urokinase, or
a variant of any of these. In some embodiments cells conjugated
with a moiety that inhibits coagulation and/or promotes
fibrinolysis may be administered to a subject at risk of blood clot
formation. For example, the subject may have an arrhythmia such as
atrial fibrillation and/or a history of pathologic coagulation
(e.g., embolism, thrombophlebitis, ischemic stroke), etc. The
moiety that inhibits coagulation and/or promotes fibrinolysis may
comprise Protein C, antithrombin, tissue factor pathway inhibitor,
or plasmin.
[0283] In some embodiments, a protein comprises a receptor or
receptor fragment (e.g., at least a portion of an extracellular
domain). In some embodiments the receptor is a cytokine receptor,
growth factor receptor, interleukin receptor, or chemokine
receptor. In certain embodiments a growth factor receptor is a
TNF.alpha. receptor (e.g., Type I TNF-.alpha. receptor), VEGF
receptor, EGF receptor, PDGF receptor, IGF receptor, NGF receptor,
or FGF receptor. In some embodiments the protein comprises at least
a sufficient portion of a receptor to bind to a natural ligand of
the receptor. In some embodiments the protein is capable of acting
as a decoy receptor, i.e., a receptor that binds a ligand and
thereby inhibits the ligand from binding to its normal receptor. In
some embodiments cells conjugated with a decoy receptor that binds
to a natural ligand are administered to a subject in order to
inhibit activity of the natural ligand. In some embodiments cells
conjugated with a decoy receptor that binds to a natural ligand are
administered to a subject suffering from a disease that can be
effectively treated by administration of a decoy receptor or other
inhibitor of the ligand and/or by administration of an inhibitor of
a natural receptor for the ligand, e.g., a disease that is at least
in part caused or exacerbated by a natural ligand of the receptor.
For example, a protein comprising a soluble TNF receptor, e.g.,
etanercept may be used in treatment of a variety of inflammatory
and autoimmune diseases such as rheumatoid arthritis, psoriasis,
ankylosing spondylitis, and Behcet's disease.
[0284] In certain embodiments, a protein comprises urate oxidase.
Urate oxidase can be formulated as a protein drug (rasburicase) for
the treatment of acute hyperuricemia, e.g., in patients receiving
chemotherapy. In some embodiments cells having urate oxidase
conjugated thereto may be administered to a subject in need of
treatment of acute hyperuricemia, e.g., a patient receiving
chemotherapy). Acute hyperuricemia may occur as a feature of tumor
lysis syndrome in patients receiving chemotherapy, e.g., for
hematologic cancers such as leukemias and lymphomas. In some
embodiments cells having urate oxidase conjugated thereto may be
administered to a subject in need of treatment of chronic
hyperuricemia, e.g., a patient with gout, e.g., gout that is
refractory to other treatments.
[0285] In some embodiments a protein is a cytokine. In some
embodiments a cytokine is an interleukin (IL) e.g., any of
IL-1-IL-38. In some embodiments a protein is a four-helix bundle
protein, e.g., a four-helix bundle cytokine. In some embodiments a
four-helix bundle cytokine is a member of the IL-2 subfamily, the
interferon (IFN) subfamily, or the IL-10 subfamily. Exemplary
four-helix bundle cytokines include, e.g., certain interferons
(e.g., a type I interferon, e.g., IFN-.alpha.), interleukins (e.g.,
IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-12), and colony stimulating
factors (e.g., G-CSF, GM-CSF, M-CSF). The IFN can be, e.g.,
interferon alpha 2a or interferon alpha 2b. See, e.g., Mott H R and
Campbell I D., Curr Opin Struct Biol. 1995, 5(1):114-21; Chaiken I
M, Williams W V, Trends Biotechnol. 1996, 14(10):369-75; Klaus W,
et al., J Mol Biol., 274(4):661-75, 1997, for further discussion of
certain of these cytokines. In some embodiments, the cytokine has a
similar structure to one or more of the afore-mentioned cytokines.
For example, the cytokine can be an IL-6 class cytokine such as
leukemia inhibitory factor (LIF) or oncostatin M. In some
embodiments, the cytokine is one that in nature binds to a receptor
that comprises a GP130 signal transducing subunit. Other four-helix
bundle proteins include growth hormone (GH) and prolactin (PRL). In
some embodiments, the protein is an erythropoiesis stimulating
agent, e.g., erythropoietin (EPO), which is also a four-helix
bundle cytokine. In some embodiments, an erythropoiesis stimulating
agent is an EPO variant, e.g., darbepoetin alfa, also termed novel
erythropoiesis stimulating protein (NESP), which is engineered to
contain five N-linked carbohydrate chains (two more than
recombinant HuEPO). In some embodiments the cytokine is one that
stimulates differentiation, activation, survival, and/or
proliferation of one or more types or subtypes of immune system
cells, e.g., T cells (e.g., CD4+ helper T cells, CD8+ cytotoxic T
cells, Tregs), NK cells, B cells, DCs, monocytes, macrophages, or
precursors of any of the foregoing.
[0286] In some embodiments, the protein comprises five helices. For
example, the protein can be an interferon beta, e.g., interferon
beta-1a or interferon beta-1b, which (as will be appreciated) is
often classified as a four-helix bundle cytokine.
[0287] In some embodiments a cytokine is an IL-12 family member.
IL-12 family members are heterodimeric cytokines and include, e.g.,
IL-12, IL-23, IL-27 and IL-35. IL-12 and IL-23 have mainly
proinflammatory properties, whereas IL-27 and IL-35 have mainly
anti-inflammatory properties. IL-12 and IL-23 share the
.beta.-chain p40 (IL-12.beta.), whereas IL-27 and IL-35 share the
.beta.-chain Epstein-Barr virus-induced gene 3 (EBI3). IL-12 and
IL-35 share the .alpha.-chain p35, whereas IL-23 and IL-27 have
unique .alpha.-chains. IL-12 and IL-23 are disulfide-linked
heterodimers whereas IL-27 and IL-35 lack a disulfide linkage.
IL-35 is produced mainly by Treg cells. IL-12, IL-23 and IL-27 are
secreted by myeloid cells such as macrophages and DCs, e.g., after
stimulation by contact with specific pathogen-associated molecular
patterns. IL-12 is composed of IL-12A (p35) and IL-12B (p40)
subunits. IL-12 is involved in the differentiation of naive T cells
into Th1 cells and plays an important role in enhancing the
activities of natural killer cells and T lymphocytes. IL-23 is a
composed of p40, which is also a component of IL-12, and p19, which
is considered the IL-23 alpha subunit. IL-23 has several
proinflammatory effects including the capacity to stimulate naive
CD4+ T cells to differentiate into Th17 cells. IL-27 consists of
two subunits p28 and EBI3. It acts as a differentiation factor in
the generation of Tr1 cells and inhibits Th17 cells. IL-35
heterodimers are composed of EBI3 and the IL-12p35 subunit. See,
e.g., Hunter, C A, Nature Reviews Immunology 5: 521-531, 2005, for
discussion of certain IL-12 family members.
[0288] In some embodiments a cytokine is IL-17. In some embodiments
a cytokine is IL-9, IL-10, IL-11, IL-13, IL-14, or IL-15.
[0289] In some embodiments, a protein comprises a biologically
active portion of a cytokine, e.g., IL-2, IL-7, IL-12, IL-15, or
IL-21. In some embodiments, a multi-subunit cytokine such as IL-12
is produced as a single polypeptide. In some embodiments the
polypeptide preserves the N-terminus of the p40 subunit of IL-12 in
a sequential p40-p35 fusion. In some embodiments the polypeptide
comprises a p35-p40 fusion. In some embodiments the subunits are
joined by a spacer, e.g. a polypeptide linker, in a fusion protein.
Examples of bioactive fusion proteins comprising the p35 and p40
subunits of IL-12, nucleic acids and vectors encoding such
proteins, and methods of producing the proteins are described in
U.S. Pat. No. 5,891,680. In some embodiments the p35 and p40 are
expressed as parts of separate polypeptides that permit
heterodimerization of the p35 and p40 subunits may be used.
[0290] In some embodiments, a protein comprises a biologically
active variant or fragment of a co-stimulatory molecule or cell
adhesion molecule, wherein the biologically variant or fragment is
capable of binding to a naturally occurring receptor, ligand, or
interaction partner of such molecule.
[0291] In some embodiments a protein is a subunit of a multisubunit
protein, e.g., a multisubunit cytokine or multisubunit cytokine
receptor. In some embodiments a subunit is unique to a particular
cytokine or cytokine receptor. In some embodiments a subunit is
found in multiple different cytokines or cytokine receptors. In
some embodiments, two or more subunits of a multisubunit protein
may be linked to form a single molecule. The two or more subunits
may be linked to form a single polypeptide, e.g., as a fusion
protein, or may be linked by any suitable linker. In some
embodiments the two or more subunits may be linked using click
chemistry. The two or more subunits may be separated from each
other by a spacer, e.g., a polypeptide spacer, which may facilitate
assembly of the subunits to form the normal quaternary structure of
the protein. In some embodiments, two or more subunits of a
multisubunit protein may be individually attached to a cell or
expressed by a cell.
[0292] In some embodiments a protein promotes survival,
proliferation and/or differentiation of one or more cell types. A
protein may provide an extracellular signal that is necessary or
promotes survival, e.g., by inhibiting apoptosis. One of ordinary
skill in the art will be aware of certain proteins that act as
survival factors for particular cell types. Such proteins include,
e.g., growth factors, cytokines, chemokines, and others.
[0293] In some embodiments, a protein comprises a growth factor for
one or more cell types. Growth factors include, e.g., members of
the vascular endothelial growth factor (VEGF, e.g., VEGF-A, VEGF-B,
VEGF-C, VEGF-D), epidermal growth factor (EGF), insulin-like growth
factor (IGF; IGF-1, IGF-2), fibroblast growth factor (FGF, e.g.,
FGF1-FGF22), platelet derived growth factor (PDGF), or nerve growth
factor (NGF) families. It will be understood that the
afore-mentioned protein families comprise multiple members. Any
member may be used in certain embodiments. In some embodiments a
growth factor promotes survival, proliferation and/or
differentiation of one or more hematopoietic cell types. For
example, a growth factor may be CSF1 (macrophage colony-stimulating
factor), CSF2 (granulocyte macrophage colony-stimulating factor,
GM-CSF), or CSF3 (granulocyte colony-stimulating factors, G-CSF),
stem cell factor (SCF), thrombopoietin (TPO), or Flt-3 ligand. In
some embodiments, mammalian cells that have a growth factor or
growth factor receptor agonist conjugated thereto may be contacted
with cells in vitro or administered to a subject in order to
promote proliferation of cells whose proliferation is stimulated by
such moieties.
[0294] In some embodiments a protein is a chemokine. Chemokines are
a family of small cytokines that have the ability to induce
directed chemotaxis in responsive cells, i.e., they are chemotactic
cytokines. Proteins may be classified as chemokines according to
shared structural characteristics such as small size (approximately
8-10 kilodaltons in size), and the presence of at least two
cysteine residues, e.g., four cysteine residues, in conserved
locations that play an important role in their 3-dimensional
structure (some chemokines contain one or more additional
cysteines). The chemokine family may be divided into four
subfamilies depending on the spacing of their first two cysteine
residues: CXC, CX3C, C (or XC), and CX3C chemokines. A chemokine
may be CCL1-CCL28, CXCL1-CXCL17, XCL1 or XCL2, or CXC3L1. Chemokine
receptors are G protein-coupled receptors containing 7
transmembrane domains. Chemokine receptors may be divided into four
families depending on the type of chemokine they bind; CXCR that
bind CXC chemokines, CCR that bind CC chemokines, CX3CR1 that binds
the CX3C chemokine (CX3CL1), and XCR1 that binds the two XC
chemokines (XCL1 and XCL2). In some embodiments, mammalian cells
that have a chemokine or chemokine receptor agonist conjugated
thereto may be contacted with cells in vitro or administered to a
subject in order to promote migration of cells whose migration is
stimulated by such moieties.
[0295] In some embodiments, a protein is a neurotrophic factor,
i.e., a factor that promotes survival, development and/or function
of neural lineage cells (which term as used herein includes neural
progenitor cells, neurons, and glial cells, e.g., astrocytes,
oligodendrocytes, microglia). For example, in some embodiments, the
protein is a factor that promotes neurite outgrowth. In some
embodiments, the protein is ciliary neurotrophic factor (CNTF; a
four-helix bundle protein) or an analog thereof such as Axokine,
which is a modified version of human ciliary neurotrophic factor
with a 15 amino acid truncation of the C terminus and two amino
acid substitutions, which is three to five times more potent than
CNTF in in vitro and in vivo assays and has improved stability
properties. In some embodiments, mammalian cells that have a
neutrophic factor or neurotrophic factor receptor agonist
conjugated thereto may be contacted with cells in vitro or
administered to a subject in order to promote survival,
development, and/or function of cells whose survival, development,
and/or function is stimulated by such moieties.
[0296] In some embodiments a protein comprises a constant domain of
an antibody (e.g., an Fc domain) that recruits Fc receptor-bearing
cells, e.g., monocytes, dendritic cells, and natural killer cells.
Upon administration of the sortagged cells to a subject, Fc
receptor-bearing cells are recruited to a location to which the
sortagged cell is present (e.g., a site of target cells in a tumor
or site of infection). The Fc receptor-bearing cells may further
promote an immune response mounted by the sortagged cells, may
promote an immune response by endogenous immune system cells of the
subject, or may mount their own immune response against target
cells. In some embodiments a protein comprises a constant domain of
an antibody (e.g., an Fc domain) that has been modified to alter
(e.g., increase or decrease) one or more activities that such Fc
domain would otherwise have. The modification may, for example,
alter the ability of the Fc domain to recruit Fc receptor-bearing
cells and/or fix complement. In some embodiments a protein does not
comprise an Fc domain or such domain is modified so that it does
not bind to an Fc receptor and/or fix complement.
[0297] In some embodiments a protein comprises or consists of a
polypeptide that is identical in sequence to or is a variant of a
naturally occurring protein or polypeptide, e.g., a variant that is
at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%
identical to a naturally occurring protein or polypeptide or
fragment thereof. In some embodiments a protein has no more than 1,
2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid differences relative to a
naturally occurring sequence. In some embodiments a naturally
occurring protein is a mammalian protein, e.g., of human origin. In
some embodiments a protein is an antibody, an antibody fragment, or
protein comprising an antigen-binding domain.
[0298] In some embodiments a protein is a chimeric protein
comprising two or more different polypeptides that are not found
joined together in nature. For example, a chimeric protein may
comprise two or more different targeting moieties or may comprise a
targeting moiety and a therapeutic agent. In some embodiments the
two or more polypeptides are joined directly to each other, e.g.,
via peptide bond(s). In some embodiments the two or more
polypeptides are joined to each other via one or more linkers,
which may comprise or consist of one or more amino acid(s), e.g., a
polypeptide. In some embodiments the two or more polypeptides are
joined in a single polypeptide chain. In some embodiments the
polypeptide chain comprises one or more amino acids, e.g., a
polypeptide linker, between any two portions of the polypeptide. If
the polypeptide comprises multiple polypeptide linkers, they may be
the same or different in sequence. Any polypeptide can be extended,
e.g., to comprise one or more additional amino acids, e.g., a
polypeptide linker. In some embodiments at least two of the
polypeptides are subunits of the same protein, e.g., the same
cytokine or cytokine receptor. In some embodiments a chimeric
protein may be generated by producing a nucleic acid that encodes
the chimeric protein and expressing the nucleic acid in a suitable
expression system using standard methods. In some embodiments
sortase may be used to generate a chimeric protein.
[0299] In some embodiments a protein is one that forms homodimers
or heterodimers, (or homo- or heterooligomers comprising more than
two subunits, such as tetramers). In certain embodiments the
homodimer, heterodimer, or oligomer structure is such that a
terminus of a first subunit is in close proximity to a terminus of
a second subunit. For example, an N-terminus of a first subunit is
in close proximity to a C-terminus of a second subunit. In certain
embodiments the homodimer, heterodimer, or oligomer structure is
such that a terminus of a first subunit and a terminus of a second
subunit are not involved in interaction with a receptor, so that
the termini can be joined via, e.g., a non-genetically encoded
peptide element, without significantly affecting biological
activity. In some embodiments, termini of two subunits of a
homodimer, heterodimer, or oligomer are conjugated via click
chemistry using a method described herein, thereby producing a
dimer (or oligomer) in which at least two subunits are covalently
joined. For example, the neurotrophins nerve growth factor (NGF);
brain-derived neurotrophic factor (BDNF); neurotrophin 3 (NT3); and
neurotrophin 4 (NT4) are dimeric molecules which share
approximately 50% sequence identity and exist in dimeric forms.
See, e.g., Robinson R C, et al., Biochemistry. 34(13):4139-46,
1995; Robinson R C, et al., Protein Sci. 8(12):2589-97, 1999, and
references therein. In some embodiments, the dimeric protein is a
cytokine, e.g., an interleukin.
[0300] In some embodiments a protein is a member of the
immunoglobulin superfamily (IgSF). The IgSF is a group of cell
surface and soluble proteins that are involved in a variety of cell
processes, such as recognition, binding, and/or adhesion. Proteins
are classified as members of the IgSF based on characteristic
shared structural features with immunoglobulins (antibodies), i.e.,
they possess a domain known as an immunoglobulin domain or fold
which is found in antibodies. Members of the IgSF include, e.g.,
antibodies, cell surface antigen receptors, co-receptors and
costimulatory molecules of the immune system, molecules involved in
antigen presentation to lymphocytes, cell adhesion molecules,
certain cytokine receptors, and various intracellular muscle
proteins. Ig domains contain about 70-110 amino acids and may be
further categorized according to their size and function. Ig
domains possess a characteristic fold formed by two sheets of
antiparallel beta strands. The sheets are held together by a
disulfide bond. Cell surface antigen receptors include (i) the T
cell receptor (TCR), which comprises two chains, either the
TCR-alpha and -beta chains or the TCR-delta and gamma chains,
associated with a CD3 complex; (ii) the B cell receptor (BCR),
which comprises cell surface immunoglobulin associated with
antigen-nonspecific signaling molecules termed Ig alpha and Ig
beta. Major histocompatibility complex (MHC) proteins are ligands
for TCRs. MHC class I proteins form a dimer with beta-2
microglobulin ((32M) and interact with the TCR on cytotoxic T
cells. MHC class II proteins have two chains (alpha and beta) that
interact with the TCR on helper T cells. MHC class I, MHC class II
and .beta.2M proteins all possess Ig domains and therefore are
members of the IgSF. MHC class I, MHC class II, and beta-2
microglobulin function as antigen presenting molecules (APMs).
Co-receptors include a variety of proteins that function together
with primary receptors to mediate a cellular event. Certain
co-receptors are expressed on the surfaces of lymphocytes and
interact with MHC molecules during TCR or BCR engagement. The
co-receptor CD4 is found on helper T cells and the co-receptor CD8
is found on cytotoxic T cells. A co-receptor complex including the
proteins CD19, CD21, and CD89 is used by the BCR. Certain
co-receptors regulate T cell function positively (e.g., CD28) or
negatively (e.g., cytotoxic T lymphocyte antigen 4 (CTLA-4)) by
interacting with distinct cell-surface receptors. Costimulatory or
inhibitory molecules include a variety of signaling receptors and
ligands that regulate the activation, expansion and effector
functions of immune system cells. A major group of IgSF
co-receptors are molecules of the CD28 family, e.g., CD28, CTLA-4,
programmed cell death-1 (PD-1), the B- and T-lymphocyte attenuator
(BTLA, CD272, also referred to as B and T cell associated), and the
inducible T-cell costimulator (ICOS, CD278). IgSF ligands of these
molecules include members of the B7 family, e.g., CD80 (B7-1), CD86
(B7-2), ICOS ligand, PD-L1 (B7-H1), PD-L2 (B7-DC), B7-H3, and
B7-H4. The leukocyte immunoglobulin-like receptors (LILR) are
members of the IgSF. They are also known as CD85, ILTs and LIR
families, and can exert immunomodulatory effects on a wide range of
immune cells.
[0301] In some embodiments, a protein is one that participates in
cell-cell or cell-substrate physical interactions. For example, the
protein may mediate binding of cells to each other or to the
extracellular matrix (ECM). A physical interaction between cells
may be between cells of the same type or between cells of different
types. In some embodiments a physical interaction between cells or
between cells and ECM may be relatively stable, such as those cell
involved in establishing or maintaining the structure or
organization of tissues. Examples of proteins that participate in
cell-cell or cell-substrate physical interactions include, e.g.,
cell adhesion molecules (CAMs). Most CAMs belong to any of four
protein families: immunoglobulin (Ig) superfamily (IgSF) CAMs,
integrins, the cadherin superfamily, and the selectins. Many CAMs
are transmembrane proteins composed of three domains: an
intracellular domain that interacts with the cytoskeleton, a
transmembrane domain, and an extracellular domain that interacts
with other CAMs or CAM ligands. Members of all CAM superfamilies
can mediate cell-cell interactions, whereas integrins can also
mediate cell-matrix interactions. CAMs bind to either the exact
same protein, known as homophilic binding, or different proteins,
called heterophilic binding. IgSF CAMs include, e.g., synaptic cell
adhesion molecules, neural cell adhesion molecules (NCAMs),
intercellular cell adhesion molecule (ICAM-1), vascular cell
adhesion molecule (VCAM-1), platelet-endothelial cell adhesion
molecule (PECAM-1). The cadherin superfamily is composed of
proteins that include extracellular cadherin domains (ECD). Such
proteins include cadherins, protocadherins, desmogleins, and
desmocollins, and others (see, e.g., Hulpiau P, van Roy F (2009),
"Molecular evolution of the cadherin superfamily". Int. J. Biochem.
Cell Biol. 41 (2): 349-69). Cadherins are Ca2+-dependent
glycoproteins. Different cadherins typically exhibit different
patterns of tissue distribution, and many of these proteins are
named according to tissue in which they are typically found, such
as epithelial (E-cadherins), placental (P-cadherins), neural
(N-cadherins), retinal (R-cadherins), brain (B-cadherins and
T-cadherins) and muscle (M-cadherins). Many cell types express
multiple cadherin types.
[0302] Integrins mediate cell interactions with a variety of
ligands on other cells or in the ECM such as collagen, fibrinogen,
fibronectin, laminin, and vitronectin. Integrins are heterodimers,
consisting of an alpha and beta subunit. There are at least 18
alpha subunits and at least 8 beta subunits in mammals, which
combine to make up at least 24 different integrin proteins. Many
integrins bind to ligands containing an RGD or LDV tripeptide.
[0303] In some embodiments a CAM or CAM ligand is conjugated to
mammalian cells using sortase. A CAM or CAM ligand may be used as a
targeting moiety. A mammalian cell that has a CAM or CAM ligand
conjugated to it may have increased ability to physically associate
with cells or ECM that comprise a compatible CAM or CAM ligand, as
compared with control cells. This property may be useful, e.g., if
cells are used in regenerative medicine or if it is desired that
the cells integrate into an organ or tissue. An appropriate CAM or
CAM ligand may be selected based on the site at which it is desired
that the cells associate or integrate. For example, a CAM or CAM
ligand known to bind to a CAM or CAM ligand present at the site may
be selected.
[0304] Selectins are a family of heterophilic CAMs that bind
fucosylated carbohydrates. Selectins are involved in a variety of
processes including constitutive lymphocyte homing and in chronic
and acute inflammation. The three selectin family members are
E-selectin (endothelial), L-selectin (leukocyte), and P-selectin
(platelet). Selectin ligands include P-selectin glycoprotein
ligand-1 (PSGL-1), which is a mucin-type glycoprotein expressed on
white blood cells. Selectin ligands include sialyl Lewis X (SLex)
(NeuAc.alpha.2-3Gal.beta.1-4(Fuc.alpha.1-3)GlcNAc) and sialyl Lewis
A (sLe(a)) tetrasaccharides and a variety of structurally similar
carbohydrate moieties, typically in the context of more extensive
binding determinants. Among other things, interaction between
selectin ligands present on endothelial cell surfaces and selectins
on leukocyte cell surfaces mediates rolling and attachment of
leukocytes to vessel walls and facilitates their extravasation into
tissues, e.g., at sites of inflammation. Selectin binding to sLe(x)
and sLe(a) present on various cancer cell types, e.g., colon,
gastric, bladder, pancreatic, breast, and prostate carcinomas, is
implicated in enhancing metastasis. In some embodiments a selectin
or selectin ligand is conjugated to mammalian cells using sortase.
In some embodiments a selectin ligand serves as a target or
targeting moiety. For example, a selectin ligand may be conjugated
to mammalian cells, e.g., immune systems cells, using sortase. The
cells may be administered to a subject, e.g., intravascularly. The
selectin may target the mammalian cells to sites of inflammation,
where endothelial cells express the cognate selectin ligand. In
some embodiments, e.g., if the inflammation is unwanted, e.g.,
associated with autoimmune disease or causing excessive symptoms,
tissue damage or cytokine release, mammalian cells that have
immunosuppressive activity (e.g., Tregs or cells that have been
stimulated to express an immunosuppressive cytokine or other
immunosuppressive molecule or modified to have an immunosuppressive
cytokine or other immunosuppressive molecule at their surface) may
be targeted to the site. In some embodiments, e.g., if the
inflammation is in response to an infection or other condition in
which it may be beneficial to stimulate an immune response,
mammalian cells that have or may stimulate appropriate effector
responses may be targeted to the site. In some embodiments
mammalian cells that have a therapeutic agent conjugated thereto
may be targeted to the site. In some embodiments a selectin serves
as a target or targeting moiety. For example, a selectin may be
conjugated to mammalian cells, e.g., immune systems cells, using
sortase. The selectin may target the mammalian cells to tumor cells
that express the cognate selectin ligand. The mammalian cells may
be immune system cells that have anti-tumor activity and/or may
have a therapeutic agent, e.g., a cytotoxic agent, at their
surface.
[0305] The invention encompasses application of the inventive
methods to any of the proteins described herein and any proteins
known to those of skill in the art. Without limitation, sequences
of certain proteins of interest are found in, e.g., U.S. Ser. Nos.
10/773,530; 11/531,531; U.S. Ser. Nos. 11/707,014; 11/429,276;
11/365,008, and/or in Table T and/or under the NCBI accession
numbers listed in Table T or encoded by genes identified by Gene ID
and/or NCBI RefSeq accession number in Table T, described by
Official Symbol (assigned by the HUGO Gene Nomenclature Committee
in the case of human genes), and/or described by name or otherwise
herein. It is noted that where multiple isoforms of a particular
protein exist, the dominant isoform, longest isoform, isoform 1, or
isoform having a particular biological activity of interest may be
selected. In some embodiments a cell-bound isoform may be selected.
In some embodiments a secreted isoform may be selected.
[0306] In some embodiments, modified versions of any protein,
wherein the modified version comprises (i) one or more nucleophilic
residues such as glycine at the N-terminus (e.g., between 1 and 10
residues) and, optionally, a cleavage recognition sequence, e.g., a
protease cleavage recognition sequence that masks the nucleophilic
residue(s); or (ii) a sortase recognition motif at or near the
C-terminus may be used in a composition or method described herein,
e.g., attachment of the modified version to a mammalian cell. In
some embodiments, the protein comprises both (i) and (ii). In some
aspects, the present disclosure provides proteins comprising any
protein described herein, e.g., any antibody, antibody fragment,
antibody chain, antibody domain, scFv, VHH, affibody, adnectin,
anticalin, cytokine, cytokine chain, Ig superfamily protein,
pro-apoptotic domain, or antigen, or a biologically active fragment
or variant of any of the foregoing; and a sequence comprising a
sortase recognition motif. In some embodiments the sortase
recognition motif is located at or near a C-terminus of the
protein.
[0307] One of skill in the art will be aware that certain proteins,
e.g., secreted eukaryotic (e.g., mammalian) proteins, often undergo
intracellular processing (e.g., cleavage of a secretion signal
prior to secretion and/or removal of other portion(s) that are not
required for biological activity), to generate a mature form. Such
mature, biologically active versions of proteins are used in
certain embodiments of the invention.
TABLE-US-00008 TABLE T Selected Protein Sequences and Selected Gene
IDs and Accession Numbers Tissue plasminogen activator (1rtf) Chain
A: TTCCGLRQY (SEQ ID NO: 5) Chain B:
IKGGLFADIASHPWQAAIFAKHHRRGGERFLCGGILISSCWILSAA
HCFQQQQQEEEEERRRRRFFFFFPPPPPPHHLTVILGRTYRVVPGE
EEQKFEVEKYIVHKEFDDDTYDNDIALLQLKSSSSSDDDDDSSSSS
SSSSSRRRRRCAQESSVVRTVCLPPADLQLPDWTECELSGYGKHE
ALSPFYSERLKEAHVRLYPSSRCTTTSSSQQQHLLNRTVTDNMLC
AGDTTTRRRSSSNNNLHDACQGDSGGPLVCLNDGRMTLVGIISW
GLGCGGQQKDVPGVYTKVTNYLDWIRDNMRP (SEQ ID NO: XX) Factor IX Chain A:
VVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCV
EETTGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNNNAAAA
AAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTTTNNNIIIFLK
FGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIY
NNMFCAGGFFHEGGGRRDSCQGDSGGPHVTEVEGTSFLTGIISW
GEECAAMMKGKYGIYTKVSRYVNWIKEKTKLT (SEQ ID NO: 6) Chain B:
MTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVP FPCGRVSVSQTSK (SEQ ID
NO: 7) Glucocerebrosidase
EFARPCIPKSFGYSSVVCVCNATYCDSFDPPALGTFSRYESTRSGR
RMELSMGPIQANHTGTGLLLTLQPEQKFQKVKGFGGAMTDAAA
LNILALSPPAQNLLLKSYFSEEGIGYNIIRVPMASCDFSIRTYTYAD
TPDDFQLHNFSLPEEDTKLKIPLIHRALQLAQRPVSLLASPWTSPT
WLKTNGAVNGKGSLKGQPGDIYHQTWARYFVKFLDAYAEHKL
QFWAVTAENEPSAGLLSGYPFQCLGFTPEHQRDFIARDLGPTLAN
STHHNVRLLMLDDQRLLLPHWAKVVLTDPEAAKYVHGIAVHW
YLDFLAPAKATLGETHRLFPNTMLFASEACVGSKFWEQSVRLGS
WDRGMQYSHSIITNLLYHVVGWTDWNLALNPEGGPNWVRNFV
DSPIIVDITKDTFYKQPMFYHLGHFSKFIPEGSQRVGLVASQKNDL
DAVALMHPDGSAVVVVLNRSSKDVPLTIKDPAVGFLETISPGYSI HTYLWHRQ (SEQ ID NO:
8) alpha galactosidase A
LDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAE
LMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGI
RQLANYVHSKGLKLGIYADVGNKTCAGFPGSFGYYDIDAQTFAD
WGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEW
PLYMWPFQKPNYTEIRQYCNHWRNFADIDDSWKSIKSILDWTSF
NQERIVDVAGPGGWNDPDMLVIGNFGLSWNQQVTQMALWAIM
AAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQG
DNFEVWERPLSGLAWAVAMINRQEIGGPRSYTIAVASLGKGVAC
NPACFITQLLPVKRKLGFYEWTSRLRSHINPTGTVLLQLENTM (SEQ ID NO: 9)
arylsulfatase-A (iduronidase, .alpha.-L-)
RPPNIVLIFADDLGYGDLGCYGHPSSTTPNLDQLAAGGLRFTDFY
VPVSLPSRAALLTGRLPVRMGMYPGVLVPSSRGGLPLEEVTVAE
VLAARGYLTGMAGKWHLGVGPEGAFLPPHQGFHRFLGIPYSHD
QGPCQNLTCFPPATPCDGGCDQGLVPIPLLANLSVEAQPPWLPGL
EARYMAFAHDLMADAQRQDRPFFLYYASHHTHYPQFSGQSFAE
RSGRGPFGDSLMELDAAVGTLMTAIGDLGLLEETLVIFTADNGPE
TMRMSRGGCSGLLRCGKGTTYEGGVREPALAFWPGHIAPGVTHE
LASSLDLLPTLAALAGAPLPNVTLDGFDLSPLLLGTGKSPRQSLFF
YPSYPDEVRGVFAVRTGKYKAHFFTQGSAHSDTTADPACHASSS
LTAHEPPLLYDLSKDPGENYNLLGATPEVLQALKQLQLLKAQLD
AAVTFGPSQVARGEDPALQICCHPGCTPRPACCHCP (SEQ ID NO: 10) arylsulfatase
B (N-acetylgalactos-amine-
SRPPHLVFLLADDLGWNDVGFHGSRIRTPHLDALAAGGVLLDNY 4-sulfatase) (1fsu)
YTQPLTPSRSQLLTGRYQIRTGLQHQIIWPCQPSCVPLDEKLLPQL
LKEAGYTTHMVGKWHLGMYRKECLPTRRGFDTYFGYLLGSEDY
YSHERCTLIDALNVTRCALDFRDGEEVATGYKNMYSTNIFTKRAI
ALITNHPPEKPLFLYLALQSVHEPLQVPEEYLKPYDFIQDKNRHH
YAGMVSLMDEAVGNVTAALKSSGLWNNTVFIFSTDNGGQTLAG
GNNWPLRGRKWSLWEGGVRGVGFVASPLLKQKGVKNRELIHIS
DWLPTLVKLARGHTNGTKPLDGFDVWKTISEGSPSPRIELLHNID
PNFVDSSPCSAFNTSVHAAIRHGNWKLLTGYPGCGYWFPPPSQY
NVSEIPSSDPPTKTLWLFDIDRDPEERHDLSREYPHIVTKLLSRLQF
YHKHSVPVYFPAQDPRCDPKATGVWGPWM (SEQ ID NO: 11) beta-hexosaminidase A
(2gjx) LWPWPQNFQTSDQRYVLYPNNFQFQYDVSSAAQPGCSVLDEAF
QRYRDLLFGTLEKNVLVVSVVTPGCNQLPTLESVENYTLTINDDQ
CLLLSETVWGALRGLETFSQLVWKSAEGTFFINKTEIEDFPRFPHR
GLLLDTSRHYLPLSSILDTLDVMAYNKLNVFHWHLVDDPSFPYES
FTFPELMRKGSYNPVTHIYTAQDVKEVIEYARLRGIRVLAEFDTP
GHTLSWGPGIPGLLTPCYSGSEPSGTFGPVNPSLNNTYEFMSTFFL
EVSSVFPDFYLHLGGDEVDFTCWKSNPEIQDFMRKKGFGEDFKQ
LESFYIQTLLDIVSSYGKGYVVWQEVFDNKVKIQPDTIIQVWREDI
PVNYMKELELVTKAGFRALLSAPWYLNRISYGPDWKDFYVVEPL
AFEGTPEQKALVIGGEACMWGEYVDNTNLVPRLWPRAGAVAER
LWSNKLTSDLTFAYERLSHFRCELLRRGVQAQPLNVGFCEQEFEQ (SEQ ID NO: 12)
Hexosaminidase A and B (2gjx) CHAIN A:
LWPWPQNFQTSDQRYVLYPNNFQFQYDVSSAAQPGCSVLDEAF
QRYRDLLFGTLEKNVLVVSVVTPGCNQLPTLESVENYTLTINDDQ
CLLLSETVWGALRGLETFSQLVWKSAEGTFFINKTEIEDFPRFPHR
GLLLDTSRHYLPLSSILDTLDVMAYNKLNVFHWHLVDDPSFPYES
FTFPELMRKGSYNPVTHIYTAQDVKEVIEYARLRGIRVLAEFDTP
GHTLSWGPGIPGLLTPCYSGSEPSGTFGPVNPSLNNTYEFMSTFFL
EVSSVFPDFYLHLGGDEVDFTCWKSNPEIQDFMRKKGFGEDFKQ
LESFYIQTLLDIVSSYGKGYVVWQEVFDNKVKIQPDTIIQVWREDI
PVNYMKELELVTKAGFRALLSAPWYLNRISYGPDWKDFYVVEPL
AFEGTPEQKALVIGGEACMWGEYVDNTNLVPRLWPRAGAVAER
LWSNKLTSDLTFAYERLSHFRCELLRRGVQAQPLNVGFCEQEFEQ (SEQ ID NO: 13) Chain
B: PALWPLPLSVKMTPNLLHLAPENFYISHSPNSTAGPSCTLLEEAFR
RYHGYIFGTQVQQLLVSITLQSECDAFPNISSDESYTLLVKEPVAV
LKANRVWGALRGLETFSQLVYQDSYGTFTINESTIIDSPRFSHRGI
LIDTSRHYLPVKIILKTLDAMAFNKFNVLHWHIVDDQSFPYQSITF
PELSNKGSYSLSHVYTPNDVRMVIEYARLRGIRVLPEFDTPGHTLS
WGKGQKDLLTPCYSDSFGPINPTLNTTYSFLTTFFKEISEVFPDQFI
HLGGDEVEFKCWESNPKIQDFMRQKGFGTDFKKLESFYIQKVLDI
IATINKGSIVWQEVFDDKAKLAPGTIVEVWKDSAYPEELSRVTAS
GFPVILSAPWYLDLISYGQDWRKYYKVEPLDFGGTQKQKQLFIG
GEACLWGEYVDATNLTPRLWPRASAVGERLWSSKDVRDMDDA
YDRLTRHRCRMVERGIAAQPLYAGYCN (SEQ ID NO: 14) Chain C:
PALWPLPLSVKMTPNLLHLAPENFYISHSPNSTAGPSCTLLEEAFR
RYHGYIFGTQVQQLLVSITLQSECDAFPNISSDESYTLLVKEPVAV
LKANRVWGALRGLETFSQLVYQDSYGTFTINESTIIDSPRFSHRGI
LIDTSRHYLPVKIILKTLDAMAFNKFNVLHWHIVDDQSFPYQSITF
PELSNKGSYSLSHVYTPNDVRMVIEYARLRGIRVLPEFDTPGHTLS
WGKGQKDLLTPCYSLDSFGPINPTLNTTYSFLTTFFKEISEVFPDQ
FIHLGGDEVEFKCWESNPKIQDFMRQKGFGTDFKKLESFYIQKVL
DIIATINKGSIVWQEVFDDKAKLAPGTIVEVWKDSAYPEELSRVT
ASGFPVILSAPWYLDLISYGQDWRKYYKVEPLDFGGTQKQKQLFI
GGEACLWGEYVDATNLTPRLWPRASAVGERLWSSKDVRDMDD
AYDRLTRHRCRMVERGIAAQPLYAGYCN (SEQ ID NO: 15) Chain D:
LWPWPQNFQTSDQRYVLYPNNFQFQYDVSSAAQPGCSVLDEAF
QRYRDLLFGTLEKNVLVVSVVTPGCNQLPTLESVENYTLTINDDQ
CLLLSETVWGALRGLETFSQLVWKSAEGTFFINKTEIEDFPRFPHR
GLLLDTSRHYLPLSSILDTLDVMAYNKLNVFHWHLVDDPSFPYES
FTFPELMRKGSYNPVTHIYTAQDVKEVIEYARLRGIRVLAEFDTP
GHTLSWGPGIPGLLTPCYSGSEPSGTFGPVNPSLNNTYEFMSTFFL
EVSSVFPDFYLHLGGDEVDFTCWKSNPEIQDFMRKKGFGEDFKQ
LESFYIQTLLDIVSSYGKGYVVWQEVFDNKVKIQPDTIIQVWREDI
PVNYMKELELVTKAGFRALLSAPWYLNRISYGPDWKDFYVVEPL
AFEGTPEQKALVIGGEACMWGEYVDNTNLVPRLWPRAGAVAER
LWSNKLTSDLTFAYERLSHFRCELLRRGVQAQPLNVGFCEQEFEQ (SEQ ID NO: 16)
phenylalanine hydroxylase (PAH) (1j8u)
VPWFPRTIQELDRFANQILSYGAELDADHPGFKDPVYRARRKQFA
DIAYNYRHGQPIPRVEYMEEEKKTWGTVFKTLKSLYKTHACYEY
NHIFPLLEKYCGFHEDNIPQLEDVSQFLQTCTGFRLRPVAGLLSSR
DFLGGLAFRVFHCTQYIRHGSKPMYTPEPDICHELLGHVPLFSDRS
FAQFSQEIGLASLGAPDEYIEKLATIYWFTVEFGLCKQGDSIKAYG
AGLLSSFGELQYCLSEKPKLLPLELEKTAIQNYTVTEFQPLYYVAE
SFNDAKEKVRNFAATIPRPFSVRYDPYTQRIEVL (SEQ ID NO: 17) Cathepsin A
APDQDEIQRLPGLAKQPSFRQYSGYLKSSGSKHLHYWFVESQKD
PENSPVVLWLNGGPGCSSLDGLLTEHGPFLVQPDGVTLEYNPYS
WNLIANVLYLESPAGVGFSYSDDKFYATNDTEVAQSNFEALQDF
FRLFPEYKNNKLFLTGESYAGIYIPTLAVLVMQDPSMNLQGLAVG
NGLSSYEQNDNSLVYFAYYHGLLGNRLWSSLQTHCCSQNKCNF
YDNKDLECVTNLQEVARIVGNSGLNIYNLYAPCAGGVPSHFRYE
KDTVVVQDLGNIFTRLPLKRMWHQALLRSGDKVRMDPPCTNTT
AASTYLNNPYVRKALNIPEQLPQWDMCNFLVNLQYRRLYRSMN
SQYLKLLSSQKYQILLYNGDVDMACNFMGDEWFVDSLNQKMEV
QRRPWLVKYGDSGEQIAGFVKEFSHIAFLTIKGAGHMVPTDKPLA AFTMFSRFLNKQPY (SEQ
ID NO: 18) G-CSF LPQSFLLKCLEQVRKIQGDGAALQEKLCATYKLCHPEELVLLGHS
LGIPWAPLLAGCLSQLHSGLFLYQGLLQALEGISPELGPTLDTLQL
DVADFATTIWQQMEELGMMPAFASAFQRRAGGVLVASHLQSFL EVSYRVLRHLA (SEQ ID NO:
19) GM-CSF EHVNAIQEARRLLNLSRDTAAEMNETVEVISEMFDLQEPTCLQTR
LELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQIITF ESFKENLKDFLLVIP (SEQ
ID NO: 20) Interferon alfa-2
CDLPQTHSLGSRRTLMLLAQMRKISLFSCLKDRHDFGFPQEEFGN
QFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQ
QLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKY
SPCAWEVVRAEIMRSFSLSTNLQESLRSKE (SEQ ID NO: 21) Interferon beta-1
MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEI
KQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLA
NVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYL
KAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN (SEQ ID NO: 22) Interferon
gamma-1b MQDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWKEESDR
KIMQSQIVSFYFKLFKNFKDDQSIQKSVETIKEDMNVKFFNSNKK
KRDDFEKLTNYSVTDLNVQRKAIDELIQVMAELGANVSGEFVKE
AENLKKYFNDNGTLFLGILKNWKEESDRKIMQSQIVSFYFKLFKN
FKDDQSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTNYSVTDL NVQRKAIHELIQVMAELSPAA
(SEQ ID NO: 23) IL-2 (1M47)
STKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKK
ATELKHLQCLEEELKPLEEVLNLAQNFHLRPRDLISNINVIVLELK
GFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 24) IL-1 (2nvh)
APVRSLNCTLRDSQQKSLVMSGPYELKALHLQGQDMEQQVVFS
MSFVQGEESNDKIPVALGLKEKNLYLSCVLKDDKPTLQLESVDP
KNYPKKKMEKRFVFNKIEINNKLEFESAQFPNWYISTSQAENMPV FLGGTKGGQDITDFTMQFVS
(SEQ ID NO: 25) TNF-alpha (4tsv)
DKPVAHVVANPQAEGQLQWSNRRANALLANGVELRDNQLVVPI
EGLFLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKS
PCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDF AESGQVYFGIIAL (SEQ ID
NO: 26) TNF-beta (lymphotoxin) (1tnr)
KPAAHLIGDPSKQNSLLWRANTDRAFLQDGFSLSNNSLLVPTSGI
YFVYSQVVFSGKAYSPKATSSPLYLAHEVQLFSSQYPFHVPLLSS
QKMVYPGLQEPWLHSMYHGAAFQLTQGDQLSTHTDGIPHLVLSP STVFFGAFAL (SEQ ID NO:
27) Erythropoietin APPRLICDSRVLERYLLEAKEAEKITTGCAEHCSLNEKITVPDTKV
NFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVKSSQPW
EPLQLHVDKAVSGLRSLTTLLRALGAQKEAISNSDAASAAPLRTI
TADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR (SEQ ID NO: 28) Insulin Chain A:
GIVEQCCTSICSLYQLENYCN (SEQ ID NO: 29) Chain B:
FVNQHLCGSHLVEALYLVCGERGFFYTPK (SEQ ID NO: 30) Growth hormone (GH)
(Somatotropin) FPTIPLSRLADNAWLRADRLNQLAFDTYQEFEEAYIPKEQIHSFW (1huw)
WNPQTSLCPSESIPTPSNKEETQQKSNLELLRISLLLIQSWLEPVQF
LRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEALLKNYG
LLYCFNKDMSKVSTYLRTVQCRSVEGSCGF (SEQ ID NO: 31) Follicle-stimulating
hormone (FSH) CHHRICHCSNRVFLCQESKVTEIPSDLPRNAIELRFVLTKLRVIQK
GAFSGFGDLEKIEISQNDVLEVIEADVFSNLPKLHEIRIEKANNLLY
INPEAFQNLPNLQYLLISNTGIKHLPDVHKIHSLQKVLLDIQDNINI
HTIERNSFVGLSFESVILWLNKNGIQEIHNCAFNGTQLDELNLSDN
NNLEELPNDVFHGASGPVILDISRTRIHSLPSYGLENLKKLRARST YNLKKLPTLE (SEQ ID
NO: 32) Leptin (1ax8)
IQKVQDDTKTLIKTIVTRINDILDFIPGLHPILTLSKMDQTLAVYQQ
ILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPEASGLETLDSL
GGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGC (SEQ ID NO: 33) Insulin-like
growth factor (or PETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDE
somatomedin) (1wqj) CCFRSCDLRRLEMYCAP (SEQ ID NO: 34) Adiponectin
(1c28) Chain A: MYRSAFSVGLETRVTVPNVPIRFTKIFYNQQNHYDGSTGKFYCNI
PGLYYFSYHITVYMKDVKVSLFKKDKAVLFTYDQYQENVDQAS
GSVLLHLEVGDQVWLQVYYADNVNDSTFTGFLLYHDT (SEQ ID NO: 35) Chain B:
MYRSAFSVGLPNVPIRFTKIFYNQQNHYDGSTGKFYCNIPGLYYF
SYHITVYMKDVKVSLFKKDKVLFTYDQYQEKVDQASGSVLLHL EVGDQVWLQVYDSTFTGFLLYHD
(SEQ ID NO: 36) Chain C:
MYRSAFSVGLETRVTVPIRFTKIFYNQQNHYDGSTGKFYCNIPGL
YYFSYHITVDVKVSLFKKDKAVLFTQASGSVLLHLEVGDQVWLQ NDSTFTGFLLYHD (SEQ ID
NO: 37) Factor VIII (aka antihemophilic factor) Chain A: (2r7e)
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSV
VYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLK
NMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGG
SHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGAL
LVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNAASARA
WPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFL
EGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQH
DGMEAYVKVDSCPEEPQFDDDNSPSFIQIRSVAKKHPKTWVHYIA
AEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMA
YTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYP
HGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPT
KSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIM
SDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNI
MHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTF
KHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGM
TALLKVSSCDKNTGDYYEDSYED (SEQ ID NO: 38) Chain B:
RSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKK
VVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRN
QASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQH
HMAPTKDEFDCKAWAYSSDVDLEKDVHSGLIGPLLVCHTNTLNP
AHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMED
PTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENI
HSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWR
VECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQ
YGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQ
GARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDS
SGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLG
MESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQV
NNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQD
GHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQS WVHQIALRMEVLGCEAQDLY
(SEQ ID NO: 39) Human serum albumin (1ao6) Chain A:
SEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEF
AKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCA
KQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK
KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP
KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFP
KAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQ
DSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKD
VCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLE
KCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKF
QNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMP
CAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALE
VDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKP
KATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQ AA (SEQ ID NO: 40)
Chain B: SEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEF
AKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCA
KQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK
KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP
KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFP
KAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQ
DSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKD
VCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLE
KCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKF
QNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMP
CAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALE
VDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKP
KATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQ AA (SEQ ID NO: 42)
Hemoglobin (1bz0) Chain A:
VLSPADKTNVKAAWGKVGAHAGEYGAEALERMFLSFPTTKTYF
PHFDLSHGSAQVKGHGKKVADALTNAVAHVDDMPNALSALSDL
HAHKLRVDPVNFKLLSHCLLVTLAAHLPAEFTPAVHASLDKFLA SVSTVLTSKYR (SEQ ID
NO: 43) Chain B: VHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFE
SFGDLSTPDAVMGNPKVKAHGKKVLGAFSDGLAHLDNLKGTFA
TLSELHCDKLHVDPENFRLLGNVLVCVLAHHFGKEFTPPVQAAY QKVVAGVANALAHKYH (SEQ
ID NO: 44) Trail (TNFSF10) Gene ID: 8743; NP_003801.1 (isoform 1);
NP_003801.1 (isoform 2); NP_001177872.1 (isoform 3) E-selectin
(SELE) Gene ID: 6401; NP_000441.2 CD3 epsilon (CD3E) Gene ID: 916;
NP_000724.1 CD3 zeta (CD247) Gene ID: 919; NP_932170.1 (isoform 1);
NP_000725.1 (isoform 2) CD3 delta (CD3D) Gene ID: 915; NP_000723.1
(isoform A); NP_001035741.1 (isoform B) CD3 gamma (CD3G) Gene ID:
917; NP_000064.1 CD28 Gene ID: NP_006130.1 (isoform 1);
NP_001230006.1 (isoform 2); NP_001230007.1 (isoform 3) Programmed
cell death 1 (PD-1) Gene ID: 5133; NP_005009.2 (PDCD1) PD-L1
(CD274) Gene ID: 29126NP_054862.1 (isoform a); NP_001254635.1
(isoform b); PD-L2 (PDCD1LG2) Gene ID: 80380; NP_079515.2 CTLA-4
Gene ID: 1493; NP_005205.2 (isoform CLTA4-TM); NP_001032720.1
(isoform CTLA4delTM) BCMA (TNFRSF17) Gene ID: 608; NP_001183.2
CD137L (TNFSF9) Gene ID: 8744; NP_003802.1
[0308] It will be appreciated that considerable structure/function
information is available regarding many of the afore-mentioned
proteins, as well as sequences from different mammalian species,
that can be used to design variants of the naturally occurring
sequence that retain significant biological activity (e.g., at
least 25%, 75%, 90%, 95%, 98%, 99%, or more of the activity of the
naturally occurring protein, or greater activity than the naturally
occurring protein). For example, crystal structures or NMR
structures of a number of proteins, in some instances in a complex
with the corresponding receptor, are available. It will be
understood that a naturally occurring sequence can be extended,
e.g., at or near its C-terminus, e.g., with a flexible peptide
spacer (e.g., any of the polypeptide linkers mentioned herein),
which may allow the polypeptide more freedom to fold and/or
interact or associate with free or cell-bound molecules or
structures in the extracellular environment after it is conjugated
to mammalian cells than would otherwise be the case. In addition,
it will be understood that, if the naturally occurring N- and
C-termini are not located in close proximity to each other in the
native structure, a naturally occurring sequence can be extended at
the N- and/or C-termini, e.g., with a flexible peptide spacer so
that the termini can come into close proximity, which may be
desirable, for example, if the polypeptide is to be
circularized.
[0309] In some embodiments, a protein has been tested in one or
more human clinical trials and, in some embodiments, demonstrated
acceptable safety in at least a Phase I trial. In some embodiments,
a protein is approved by the US Food & Drug Administration (or
an equivalent regulatory authority such as the European Medicines
Evaluation Agency) for use in treating a disease or disorder in
humans. In some embodiments a protein is a PEGylated version of the
protein.
[0310] In some embodiments an agent conjugated to a living
mammalian cell using sortase comprises an antigen or a binding
moiety that binds to an antigen. In some embodiments an antigen is
any molecule or complex comprising at least one epitope recognized
by a B cell, e.g., a mammalian or avian B cell and/or by a T cell,
e.g., a mammalian or avian T cell. An antigen may comprise a
polypeptide, a polysaccharide, a carbohydrate, a lipid, a nucleic
acid, or combination thereof. In some embodiments an antigen
comprises a protein, e.g., a polypeptide encoded or expressed by an
organism. A polypeptide antigen may comprise or consist of a full
length polypeptide or a portion thereof, such as a peptide at least
about 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids long. In
some embodiments an antigen comprises a lipid or glycolipid such as
.alpha.-galactosylceramide (.alpha.-GalCer), which is recognized by
iNKT cells. An antigen may be naturally occurring or synthetic. In
some embodiments, an antigen is naturally produced by and/or is
genetically encoded by a pathogen, an infected cell, or a
neoplastic cell (e.g., a cancer cell). In some embodiments, an
antigen is an autoantigen ("self antigen"), or an agent that has
the capacity to initiate or enhance an autoimmune response. In some
embodiments, an antigen is a graft-associated antigen. In some
embodiments, an antigen is produced or genetically encoded by a
virus, bacteria, fungus, or parasite which, in some embodiments, is
a pathogenic agent. In some embodiments, an agent (e.g., virus,
bacterium, fungus, parasite) infects and, in some embodiments,
causes disease in, at least one mammalian or avian species, e.g.,
human, non-human primate, bovine, ovine, equine, caprine, and/or
porcine species. In some embodiments, a pathogen is intracellular
during at least part of its life cycle. In some embodiments, a
pathogen is extracellular. In some embodiments an antigen comprises
a molecule that is produced by an infected cell as a result of
infection by a pathogen. In some embodiments, an antigen is an
envelope protein, capsid protein, secreted protein, structural
protein, cell wall protein or polysaccharide, capsule protein or
polysaccharide, or enzyme. In some embodiments an antigen is a
toxin, e.g., a bacterial toxin.
[0311] In some embodiments mammalian cells, e.g., hematopoietic
cells, e.g., immune system cells or red blood cells, that have an
epitope, antigen or portion thereof conjugated thereto by sortase
may be used as vaccine components. As used herein, "vaccine" refers
to a product or composition that may be administered to a subject
to modulate the subject's immune system or immune response towards
one or more entit(ies) of interest. A vaccine often contains an
agent that resembles at least a portion of an entity against which
an immune response is desired, e.g., a disease-causing
microorganism, parasite, or toxin, or an agent that resembles an
entity against which an immune response is not desired, e.g., a
self antigen or environmental allergen. In some embodiments an
agent stimulates the body's immune system to recognize an entity as
foreign, destroy it, and "remember" it (e.g., by inducing formation
of memory T and/or B cells), so that the immune system can more
easily recognize and destroy such an entity that it subsequently
encounters. In some embodiments an agent stimulates the body's
immune system to recognize an entity as "self" or not to recognize
the entity, so that the immune system does not mount a response
against the entity. For example, it may be desirable to inhibit an
immune response towards self antigens or graft-associated antigens.
A vaccine may be prophylactic (e.g., to prevent or reduce the
severity of a future infection by a pathogen or exposure to an
allergen), or therapeutic (e.g., vaccines against cancer or to
treat autoimmune disease or an existing allergy). In some
embodiments a vaccine modulates the adaptive immune system or a
component thereof. In some embodiments a vaccine is designed to
modulate the immune response towards a single antigen,
microorganism, or other entity. In some embodiments a vaccine is
designed to modulate the immune response towards two or more
strains of the same microorganism or entity, two or more
microorganisms or entities, or two or more distinct antigens of a
tumor, graft, or self cell or structure. Sortagged cells used as a
vaccine or vaccine component may be used to deliver an epitope,
antigen or portion thereof to a subject in order to modulate an
immune response of the subject towards an entity that comprises the
epitope or antigen. In some embodiments an antigen conjugated to
animal cells using sortase may be any antigen used in a
conventional vaccine known in the art.
[0312] One of ordinary skill in the art will be aware of numerous
microbes (e.g., viruses, bacteria, fungi, protozoa) and
multicellular parasites from which antigens or epitopes may be
derived, e.g., microbes and parasites capable of causing disease in
mammals. In some embodiments an antigen is a surface protein or
polysaccharide of, e.g., a viral capsid, envelope, or coat, or
bacterial, fungal, protozoal, or parasite cell. In some embodiments
an antigen is a toxin, e.g., a toxin produced by a bacterium. A
toxin may be provided in an inactivated form, e.g., as a toxoid. An
antigen or epitope may be modified, e.g., by chemical treatment
(e.g., formaldehyde) or physical treatment (e.g., heat) and/or by
conjugation with a second agent. It will be understood that an
antigen, e.g., a protein, "derived from" a particular microbe or
parasite can be produced using any suitable method, e.g., using
recombinant DNA technology in yeast, bacteria, or cell cultures. In
some embodiments a variant antigen may be used. For example, a
native sequence may be modified to render it more immunogenic. In
some embodiments an antigen or epitope is sufficiently similar to a
naturally occurring antigen or epitope such that it binds with at
least about 10%, 20%, 30%, least 50%, 60%, 70%, 80%, 90%, 95%, or
the same affinity to an antigen receptor or antibody that binds to
the naturally occurring antigen or epitope. In some embodiments an
antigen or epitope is sufficiently similar to a naturally occurring
antigen or epitope to elicit a desired response.
[0313] Exemplary viruses include, e.g., Retroviridae (e.g.,
lentiviruses such as human immunodeficiency viruses, such as
HIV-I); Caliciviridae (e.g. strains that cause gastroenteritis);
Togaviridae (e.g. equine encephalitis viruses, rubella viruses);
Flaviridae (e.g. dengue viruses, encephalitis viruses, yellow fever
viruses, hepatitis C virus); Coronaviridae (e.g. coronaviruses);
Rhabdoviridae (e.g. vesicular stomatitis viruses, rabies viruses);
Filoviridae (e.g. Ebola viruses); Paramyxoviridae (e.g.
parainfluenza viruses, mumps virus, measles virus, respiratory
syncytial virus); Orthomyxoviridae (e.g. influenza viruses);
Bunyaviridae (e.g. Hantaan viruses, bunga viruses, phleboviruses
and Nairo viruses); Arenaviridae (hemorrhagic fever viruses);
Reoviridae (erg., reoviruses, orbiviurses and rotaviruses);
Birnaviridae; Hepadnaviridae (Hepatitis B or C virus); Parvoviridae
(parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses);
Adenoviridae; Herpesviridae (herpes simplex virus (HSV) 1 and 2,
varicella zoster virus, cytomegalovirus (CMV), EBV, KSV);
Poxviridae (variola viruses, vaccinia viruses, pox viruses); and
Picornaviridae (e.g. polio viruses, hepatitis A virus;
enteroviruses, human coxsackie viruses, rhinoviruses,
echoviruses).
[0314] Bacteria include, e.g., gram positive, gram negative, and
acid-fast bacteria. Bacteria may be cocci, rod-shaped, spirochetes.
Exemplary bacteria include, e.g., Helicobacter pylori, Borellia
(e.g., B. burgdorferi, B. afzelii, B. garinii), Legionella
pneumophilia, Mycobacteria (e.g., M. tuberculosis, M. avium, M,
intracellulare, M. kansasii, M. gordonae), Staphylococcus (e.g.,
Staphylococcus aureus), Neisseria gonorrhoeae, Neisseria
meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group
A Streptococcus), Streptococcus agalactiae (Group B Streptococcus),
Streptococcus (viridans group), Streptococcus faecalis,
Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcus
pneumoniae, Campylobacter sp., Enterococcus sp., Chlamydia sp.,
Haemophilus influenzae, Bordetella (e.g., B. pertussis, B.
parapertussis, B. bronchiseptica), Bacillus anthracis,
Corynebacterium diphtheriae, Erysipelothrix rhusiopathiae,
Clostridia (e.g., Clostridium perfringens, Clostridium tetani,
Clostridium difficile), Enterobacter aerogenes, Klebsiella
pneumoniae, Pasteurella multocida, Bacteroides sp., Fusobacterium
nucleatum, Streptobacillus moniliformis, Treponema pallidum,
Treponema pertenue, Leptospira, Actinomyces israelii and
Francisella tularensis, E. coli (e.g., pathogenic E. coli).
[0315] In some embodiments a fungus is a member of the phylum
Ascomycota, Basidiomycota, Chytridiomycota, Glomeromycota, or
Zygomycota. The fungus may be a member of a genus selected from the
group consisting of Aspergillus, Blastomyces, Candida,
Coccidioides, Cryptococcus, Epidermophytum, Exserohilum, Fusarium,
Histoplasma, Malassezia, Microsporum, Mucor, Paracoccidioides,
Penicillium, Pichia, Pneumocystis, Pseudallescheria, Rhizopus,
Rhodotorula, Scedosporium, Schizophyllum, Sporothrix, Stachybotrys,
Saccharomyces, Trichophyton, Trichosporon, Bipolaris, Exserohilum,
Curvularia, Alternaria, or Cladophialophora. Exemplary fungi
include, e.g., Aspergillus, such as Aspergillus flavus, Aspergillus
fumigatus, Aspergillus niger, Aspergillus clavatus, Blastomyces,
such as Blastomyces dermatitidis, Candida, such as Candida
albicans, Candida glabrata, Candida guilliermondii, Candida krusei,
Candida parapsilosis, Candida tropicalis, Coccidioides, such as
Coccidioides immitis, Cryptococcus, such as Cryptococcus
neoformans, Epidermophyton, Fusarium, Histoplasma, such as
Histoplasma capsulatum, Malassezia, such as Malassezia furfur,
Microsporum, Mucor, Paracoccidioides, such as Paracoccidioides
brasiliensis, Penicillium, such as Penicillium marneffei, Pichia,
such as Pichia anomala, Pichia guilliermondii, Pneumocystis, such
as Pneumocystis carinii, Pseudallescheria, such as Pseudallescheria
boydii, Rhizopus, such as Rhizopus oryzae, Rhodotorula, such as
Rhodotorula rubra, Scedosporium, such as Scedosporium apiospermum,
Schizophyllum, such as Schizophyllum commune, Sporothrix, such as
Sporothrix schenckii, Trichophyton, such as Trichophyton
mentagrophytes, Trichophyton rubrum, Trichophyton verrucosum,
Trichophyton violaceutn, Trichosporon, such as Trichosporon asahii,
Trichosporon cutaneum, Trichosporon inkin, and Trichosporon
mucoides. In some embodiments a fungus is Coccidioides immitis,
Coccidioides posadasii. Cryptococcus neoformans, C. gattii, C.
albidus, C. laurentii, C. uniguttulas, E. floccosum, Fusarium
graminearum, Fusarium oxysporum fsp. cubense, a member of the
Fusarium solani complex, Fusarium oxysporum, Fusarium
verticillioides, Fusarium proliferatum, Malassezia furfur, Mucor
circinelloides, Paracoccidioides brasiliensis, Penicillium
marneffei, Pichia anomala, Pichia guilliermondi, Pneumocystis
carinii, Pneumocystis jirovecii, Pseudallescheria boydii, Rhizopus
oryzae, Rhodotorula rubra, Scedosporium apiospermum, Schizophyllum
commune, Sporothrix schenckii, Trichophyton mentagrophytes,
Trichophyton rubrum, Trichophyton verrucosum, Trichophyton
tonsurans, or Trichophyton violaceum, Trichosporon asahii,
Trichosporon cutaneum, Trichosporon inkin, Trichosporon mucoides,
Exserohilum rostratum E. meginnisii, or E. longirostratum.
[0316] In some embodiments a parasite is a protozoan. In some
embodiments the parasite belongs to the phylum Apicomplexa.
Exemplary parasites include, e.g., parasites of the genus
Plasmodium (Plasmodium falciparum, Plasmodium vivax, Plasmodium
ovale curtisi, Plasmodium ovale wallikeri, Plasmodium malariae, or
Plasmodium knowlesi), Trypanosoma, Toxoplasma (e.g., Toxoplasma
gondii), Leishmania (e.g., Leishmania major), Isospora,
Schistosoma, or Cryptosporidium. In some embodiments a member of
the genus Cryptosporidium is C. parvum, C. hominis, C. canis, C.
felis, C. meleagridis, or C. muris. In some embodiments a member of
the genus Isospora is Isospora belli. In some embodiments a member
of the genus Babesia is Babesia microti or Babesia divergens.
[0317] In some embodiments a protozoan is a member of a genus of
amoebae. In some embodiments a protozoan is a member of the genus
Entamoeba. In some embodiments a member of the genus Entamoeba is
Entamoeba histolytica, Entamoeba dispar, or Entamoeba moshkovskii.
In some embodiments a protozoan is a member of the genus Naegleria,
e.g., Naegleria fowleri. In some embodiments a protozoan is a
member of the genus Balamuthia, e.g., Balamuthia mandriallaris. In
some embodiments a protozoan is a member of the genus Acanthameba.
In some embodiments a member of the genus Giardia is Giardia
lamblia. In certain embodiments a protozoon is a member of genus
Sarcocystis, e.g., Sarcocystis bovohominis, Sarcocystis suihominis
or Sarcocystis bovicanis. In certain embodiments, a protozoon is a
member of genus Cyclospora, e.g., Cyclospora cayetanensis. In
certain embodiments, a protozoan is a member of genus Neospora,
e.g., Neospora caninum. In certain embodiments, a protozoan is a
member of genus Theileria e.g., Theileria parva. In certain
embodiments, a protozoan is a member of genus Trichomonas, e.g.,
Trichomonas vaginalis. In some embodiments a protozoan is a
kinetoplastid. In some embodiments a kinetoplastid is a
trypanosomatid, e.g., a member of the genus Leishmania, e.g., L.
donovani, L, major, L, tropica, or L. braziliensis, or a member of
the genus Trypanosoma, e.g., T, brucii, T. cruzii, T. congolense,
or T. equiperdum.
[0318] In some embodiments a parasite resides extracellularly
during at least part of its life cycle. Examples include nematodes,
trematodes (flukes), and cestodes. In some embodiments an antigen
may be from a nematode such as Ascaris, Enterobius, Thichuris,
and/or cestodes such as Taenia, Hymenolepis, and Echinococcus, a
cestode such as Taenia, Hymenolepis, Echinococcus, or Fasciola, a
trematode such as Schistosoma. In some embodiments an antigen is
from Trichinella, Diphyllobothrium, Clonorchis, Paragonimus,
Ancylostoma, Necator, Strongyloides, Wuchereria, Onchocerca, or
Dracunculus. In some embodiments an antigen is from an intestinal
helminth. In various embodiments an antigen can originate from any
component of the parasite or can be derived from parasites at any
stage of their life cycle of the parasite, e.g., any stage that
occurs within an infected organism such as a mammalian or avian
organism. In some embodiments an antigen is derived from eggs of
the parasite, cysts, or substances secreted by the parasite.
[0319] A graft-associated antigen may be any antigen expressed by
or present in a transplanted tissue, organ, or cells. In some
embodiments a graft-associated antigen is at least in part exposed
at the surface of transplanted cells. For example, a
graft-associated antigen may be a cell-surface protein expressed by
transplanted cells. In some embodiments a graft-associated antigen
is present in transplanted tissue, organ, or cells, but is absent
or substantially absent (e.g., not detectable using standard
detection methods) in a subject (recipient) that receives the
transplanted tissue, organ, or cells. In some embodiments a
graft-associated antigen is expressed by transplanted cells but is
not expressed at a detectable level by receipient cells of the same
cell type as the transplanted cells or, in some embodiments. In
some embodiments a graft is from a donor of a different species to
the recipient (i.e., the graft is a xenograft), in which case many
proteins expressed by the transplanted cells may be
graft-associated antigens. In some embodiments a graft-associated
antigen is a polypeptide that is polymorphic within the particular
species to which the subject belongs. For example, human leukocyte
antigens (HLA) antigens, e.g., major histocompatibility antigens
class I (MHC I) and class II (MHCII) are highly polymorphic.
[0320] In some embodiments an antigen is a tumor antigen (TA). In
general, a tumor antigen can be any antigenic substance produced by
cells in a tumor, e.g., tumor cells or in some embodiments tumor
stromal cells (e.g., tumor-associated cells such as
cancer-associated fibroblasts or tumor-associated vasculature). In
many embodiments, a TA is a molecule (or portion thereof) that is
differentially expressed by tumor cells as compared with non-tumor
cells. A TA may be expressed by a subset of tumors of a particular
type and/or by a subset of cells in a tumor. A TA may at least in
part exposed at the cell surface of tumor cells or tumor stromal
cells. In some embodiments a TA comprises an abnormally modified
protein, lipid, glycoprotein, or glycolipid. Tumor antigens may
include, e.g., proteins that are normally produced in very small
quantities and are expressed in larger quantities by tumor cells,
proteins that are normally produced only in certain stages of
development, proteins whose structure (e.g., sequence or
post-translational modification(s)) is modified due to mutation in
tumor cells, or normal proteins that are (under normal conditions)
sequestered from the immune system. In some embodiments, a TA is an
expression product of a mutated gene, e.g., an oncogene or mutated
tumor suppressor gene, an overexpressed or aberrantly expressed
cellular protein, an antigen encoded by an oncogenic virus (e.g.,
HBV; HCV; herpesvirus family members such as EBV, KSV; papilloma
virus, etc.), or an oncofetal antigen. Oncofetal antigens are
normally produced in the early stages of embryonic development and
largely or completely disappear by the time the immune system is
fully developed. Examples are alphafetoprotein (AFP, found, e.g.,
in germ cell tumors and hepatocellular carcinoma) and
carcinoembryonic antigen (CEA, found, e.g., in bowel cancers and
occasionally lung or breast cancer). Tyrosinase is an example of a
protein normally produced in very low quantities but whose
production is greatly increased in certain tumor cells (e.g.,
melanoma cells). Other exemplary TAs include, e.g., CA-125 (found,
e.g., in ovarian cancer); MUC-1 (found, e.g., in breast cancer,
ovarian cancer, and others); HER-2/neu (found, e.g., in breast
cancer); melanoma-associated antigen (MAGE; found, e.g., in
malignant melanoma); prostatic acid phosphatase (PAP, found in
prostate cancer), Wilms' tumor 1 protein (WT1, a transcription
factor overexpressed in malignant mesothelioma, leukemias, and
other solid tumors); CO17-1A (found, e.g., in colon cancer), GD2 (a
disialoganglioside expressed on tumors of neuroectodermal origin,
including human neuroblastoma and melanoma), epithelial cell
adhesion molecule (Epcam; epithelial tumors). In some embodiments a
TA is a cancer/testis (CT) antigen. CT antigens are a family of
proteins that are frequently expressed in a large variety of
malignancies but are generally absent from healthy tissue, except
for the testis and placenta. CT antigens include NY-ESO-1 and
LAGE-1. In some embodiments a tumor antigen comprises human
telomerase reverse transcriptase (hTERT). hTERT is a protein of
1132 amino acid residues and is broadly expressed in cancers but
exhibits little or no expression in most normal somatic cells. In
some embodiments a TA is an NKG2D ligand such as MICA, MICB, or
ULBP1-6.
[0321] In some embodiments an antigen may be a protein that is
found on normal B cells and plasma cells such as CD19 or CD20.
These proteins may be useful as a target in subjects with
hematologic malignancies involving such cells, such as various B
cell malignancies, e.g., B cell lymphomas, acute lymphoblastic
leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell
leukemias (HCL), acute myelogenous leukemia (AML), and multiple
myeloma (MM). Other useful protein targets in hematologic
malignancies include CD22 in, e.g., HCL and ALL (e.g., B-ALL), CD30
in, e.g., Hodgkins lymphoma and anaplastic large cell lymphoma,
CD37 in, e.g., CLL, and CD38 in, e.g., MM.
[0322] In some embodiments a tumor antigen is mesothelin (Gene ID:
10232 (MSLN; the gene encodes a precursor protein that is cleaved
into two products, megakaryocyte potentiating factor and
mesothelin); NP_001170826.1 (isoform 1); NP_037536.2 (isoform 2).
Mesothelin is a glycosylphosphatidylinositol (GPI) anchored cell
surface protein that is highly expressed in a variety of cancer
such as mesothelioma, ovarian cancer, pancreatic cancer, and is
also expressed in lung adenocarcinoma, uterine serous carcinoma,
cholangiocarcinoma, squamous cell carcinoma, and acute myeloid
leukemia (Tang, Z, et al., Anticancer Agents Med Chem. 2013 Feb. 1;
13(2): 276-280, and references therein). Mesothelin can bind to
MUC16 (also known as CA-125), to mediate heterotypic cell adhesion.
A variety of agents that bind to mesothelin are known in the art.
Such agents, or others, may be used as binding moieties or
targeting moieties. For example, MORAb-009 (amatuximab), is a
chimeric monoclonal antibody containing a single chain murine
variable region (scFv) (murine anti-mesothelin scFv SS1) and human
IgG.gamma.1 and k constant regions. A human mAb, m912 that
specifically binds to cell surface associated mesothelin was
isolated from a human Fab library (Feng Y, et al., Mol. Cancer
Ther. 2009; 8:1113-1118). A high-affinity human mAb named HN1 was
identified based on a scFv isolated by phage display technology (Ho
M, et al., Int. J. Cancer. 2011; 128:2020-2030). In some
embodiments mesothelin or a fragment or variant thereof that binds
to CA-125 may be used as a binding moiety, e.g., to target cells to
tumor cells that expess CA-125. In some embodiments the fragment of
mesothelin comprises at least amino acids 296-359, consisting of 64
amino acids at the N-terminal of cell surface mesothelin.
[0323] In some embodiments a tumor antigen is a glypican, e.g.,
glypican 3 (GPC3). Glypicans are GPI-anchored proteins expressed by
a variety of different cell types. Humans have six glypican
proteins; GPC1-GPC6. Glypican 3 (GPC3) is expressed at high levels
in certain tumors of various types, including hepatocellular
carcinoma, colorectal cancer, ovarian clear cell carcinoma (CCC),
and melanoma. Glypican 1 (GPC1) is expressed at high levels in a
number of tumor types, including certain breast cancers and
pancreatic cancer (e.g., pancreatic ductal adenocarcinoma). A
variety of agents that bind to glypicans are known in the art. Such
agents, or others, may be used as targeting moieties. For example.
In some embodiments a subject with a tumor that has increased GPC3
expression has an elevated level of a soluble GPC3 fragment in
their blood as compared with a normal level, as a result of
cleavage of cell surface GPC3. In some embodiments the binding
moiety binds to an epitope that comprises a portion of the
extracellular portion of the C-terminal domain of GPC3 (e.g.,
within the C-terminal .about.30 kD up to about amino acid 560) so
as to bind to GPC3 molecules that remain attached to the cell
surface. A variety of antibodies that bind to GPC3 are known in the
art. Examples include monoclonal antibodies 1G12 (Capurro M, et al.
Gastroenterology. 2003; 125:89-97) and YP6, YP7, YP8, YP9 and YP9.1
(Phung, Y., et al., MAbs. 2012 Sep. 1; 4(5): 592-599) A humanized
and stabilized version of the murine anti-GCP3 monoclonal antibody
GC33 has been described (Nakano, K., et al, Anticancer Drugs. 2010;
21:907-16). HN3 is a human heavy-chain variable domain antibody
with high affinity (Kd=0.6 nM) for cell-surface-associated GPC3
molecules that recognizes a conformational epitope that requires
both the amino and carboxy terminal domains of GPC3 (Feng, M., et
al., Proc Natl Acad Sci USA. 2013; 110(12):E1083-91).
[0324] In some embodiments an antigen is chondroitin sulfate
proteoglycan-4 (CSPG4). CSPG4 is highly expressed in melanoma,
breast cancer (including triple negative breast cancer), head and
neck squamous cell carcinoma, mesothelioma, glioblastoma, clear
cell renal carcinoma, and sarcomas (Wang X, et al. Curr Mol Med.
2010 June; 10(4):419-29). A CSPG4-specific fully human single-chain
antibody termed scFv-FcC21 has been described (Wang, X., et al.,
Cancer Res. (2011), 71(24):7410-22). scFv-FcC21 or its
antigen-binding domain may be used to target cells to tumors.
[0325] In some embodiments an antigen may be a signaling lymphocyte
activation molecule (SLAM) family receptor, such as SLAM or SLAMf7
(also known as CS1 and CD319).
[0326] In some embodiments an antigen is B cell maturation factor
(BCMA), also known as B cell maturation antigen (BCMA, also known
as CD269 and TNFRSF17). BCMA is a tumor necrosis family receptor
(TNFR) member that is expressed in cells of the B cell lineage,
such as terminally differentiated B cells and plasma cells. BCMA
delivers pro-survival cell signals upon binding of its ligands, B
cell activator of the TNF family (BAFF) and a proliferation
inducing ligand (APRIL). Among other things, BCMA has functional
activity in mediating the survival of B lineage cells such as
plasma cells that maintain long-term humoral immunity. The
expression of BCMA has also been linked to a number of cancers,
autoimmune disorders, and infectious diseases. In some embodiments,
cells are conjugated with a binding moiety that binds to BCMA. In
some embodiments, cells conjugated with a binding moiety that binds
to BCMA are used to deplete or inhibit a biological activity of
cells that express BCMA. An exemplary binding moiety is SG1, an
antibody that binds to BCMA and inhibits its activity (Ryan, M C,
Mol Cancer Ther. 2007; 6(11):3009-18). In some embodiments, cells
that are conjugated with a moiety that binds to BCMA are cytotoxic
immune cells, e.g., cytotoxic T cells or NK cells and/or may have a
cytotoxic moiety, e.g., a pro-apoptotic moiety such as TRAIL,
attached thereto. The moiety that binds to BCMA, the cytotoxic
moiety, or both, may be attached to the cell, e.g., to a
non-genetically engineered endogenous polypeptide expressed by the
cell, using sortase. In some embodiments the cells that express
BCMA are abnormally reactive and/or autoantibody-secreting plasma
cells and/or B cells. Depletion or inhibition of such cells may be
useful in the treatment of a wide variety of autoimmune diseases,
such as systemic lupus erythematosus (SLE), rheumatoid arthritis
(RA), Sjogren's syndrome, or other autoimmune diseases. In some
embodiments, cells conjugated with a moiety that binds to BCMA are
used to deplete cancer cells that express BCMA. BCMA is expressed
on a number of cancers, including a variety of hematologic
malignancies such as Hodgkin's and non-Hodgkin's lymphomas and
multiple myeloma, and its biological relevance in maintaining the
viability and proliferation of various malignant cells has been
demonstrated (see, e.g., Chiu, A, et al., Blood. 2007 Jan. 15;
109(2):729-39). BCMA expression has also been found on a variety of
other tumor types such as glioblastoma, leukemia, Waldenstrom
macroglobulinemia, and glioblastomas.
[0327] In some embodiments, an antigen is CD19. In some
embodiments, cells are conjugated with a moiety that binds to CD19.
In some embodiments, the binding moiety comprises an antibody that
binds to CD19. Examples of such antibodies include XmAb5603 or
XmAb5574 (Xenocor, Inc., Monrovia, Calif. and Morphosys, AG,
Martinsried, Germany), which are IgG1, humanized MAbs (Horton H M,
et al, Cancer Res 2008; 68(19):8049-8057). In some embodiments the
murine monoclonal antibody FMC63 or a humanized version thereof is
used as a binding moiety that binds to CD19. In certain embodiments
anti-CD19 monoclonal antibodies described in U.S. Pat. Pub. No.
20110104150 may be used.
[0328] In some embodiments a TA is expressed by tumor-associated
stromal cells (e.g., tumor-associated fibroblasts or
tumor-associated macrophages) or by tumor-associated vasculature.
In some embodiments a TA is a component of the modified
subendothelial tumor extracellular matrix. Such component(s) may be
secreted by tumor cells or tumor-associated cells. Examples include
certain splice isoforms of fibronectin or of tenascin-C.
Fibronectin is a large glycoprotein found in the extracellular
matrix of mammalian tissues and plasma. Under tissue remodeling
conditions, alternative splicing can lead to the insertion of EDB,
an extra 91-amino-acid type III homology domain, into fibronectin.
EDB is typically undetectable in healthy individuals, but in many
aggressive solid tumors EDB is highly expressed around tumor
vasculature. L19 is an antibody that recognizes EDB with high
affinity and has been shown to localize to tumor blood vessels in
animal models and cancer patients. Tenascins are glycoproteins
found in the extracellular matrix of vertebrates. Isoforms of
tenascin can arise in tumors through alternative splicing at sites
of neo-angiogenesis. The C domain of tenascin is undetectable in
normal adult tissue but strongly expressed in a perivascular
pattern in brain and lung tumors. The F16 antibody recognizes the
extra-domain A1 of tenascin and has shown selective accumulation at
tumors and sites of inflammation in inflammatory disorders in
animals and humans (see List, T. and Neri, D. Clinical
Pharmacology: Advances and Applications 2013:5 (Suppl 1) 29-45, and
references therein, for discussion of these and other antibodies
that bind to the same antigens or other antigens of interest).
[0329] In some embodiments, an antigen is a molecular component
(eg, histones or DNA) that may be released at sites of cell death,
such as necrotic areas in tumors. Binding moieties, e.g.,
antibodies, that bind to such antigens may be used to target cells
to a tumor. For example, human NHS76 is a phage display derived
human monoclonal antibody that recognizes nucleic acids exposed by
necrotic tumor cells as well as metastases (Sharifi J, et al.
Hybrid Hybridomics. 2001; 20(5-6):305-312).
[0330] In some embodiments an antigen comprises a peptide. In some
embodiments the peptide is at least 6, 7, 8, 9, 10, 11, 12, 13, 14,
or 15 amino acids long. In some embodiments the peptide is between
20 and 50 amino acids long. In some embodiments the peptide is
between 8 and 30, between 15 and 25, between 20 and 30, between 25
and 35, or between 35 and 50 amino acids long. Peptides may bind
directly to MHC molecules expressed on cell surfaces, may be
ingested and processed by APC and displayed on APC cell surfaces in
association with MHC molecules, and/or may bind to purified MHC
proteins (e.g., MHC oligomers). In some embodiments a peptide
contains at least one epitope capable of binding to an appropriate
MHC class I protein and/or at least one epitope capable of binding
to an appropriate MHC class II protein. In some embodiments a
peptide comprises a CTL epitope (e.g., the peptide can be
recognized by CTLs when bound to an appropriate MHC class I
protein). In some embodiments a peptide comprises a Th epitope
(e.g., the peptide can be recognized by Th cells when bound to an
appropriate MHC class II protein). In some embodiments the sequence
of a peptide comprises or consists of the sequence of a portion of
a longer polypeptide that is naturally encoded by a pathogen or a
neoplastic cell or is produced by an infected cell as a result of
the infection. In some embodiments an antigen is an artificial
polypeptide whose sequence comprises multiple distinct sequences
from different distinct polypeptides. For example, sequence of
peptides that would be found as portions of distinct antigens in
nature may be combined to produce a composite antigen comprising
epitopes originating from such distinct antigens. For example, an
antigen may comprise a polypeptide represented as X1-X2 . . . -Xn,
where X1, X2 . . . Xn represent peptides found in distinct
proteins, and in which n may range, e.g., from 2 to 5, 10, 20, or
more. It will be understood that X1, X2, etc., may be directly
adjacent to each other or joined by intervening linker(s). The
resulting composite antigen may be capable of stimulating an immune
response to multiple distinct antigens, e.g., each of the distinct
antigens. In some embodiments multiple epitopes, e.g., multiple
immunodominant epitopes, are combined to generate a composite
antigen. In some embodiments the sequence of an antigen comprises
multiple distinct variants of a polypeptide, wherein such variants
are found in different strains, serotypes, or subtypes of a
pathogen. For example, an antigen may comprise peptides or
polysaccharides obtained from at least 2, 5, 10, 20, or more
strains, serotypes, or subtypes (e.g., clades) of a pathogen. In
some embodiments the sequence of an antigen comprises multiple
distinct variants of a polypeptide, wherein such variants are found
in different pathogenic strains or different pathogenic species
belonging to a particular genus. In some embodiments at least some
of the different polypeptides are naturally encoded by the same
pathogen. In some embodiments the different polypeptides are
naturally encoded by different pathogens. In some embodiments the
different pathogens are viruses, bacteria, fungi, or parasites. In
some embodiments the sequence of an antigen comprises multiple
distinct sequences from different distinct tumor antigens. In some
embodiments an antigen is any antigen known or used in the art as a
vaccine or vaccine component. In some embodiments an epitope or
antigen is a synthetic compound whose sequence or structure
resembles that of a naturally occurring epitope antigen. For
example, in some embodiments the sequence of a naturally occurring
epitope or antigen may be altered by addition, deletion, or
substitution of one or more amino acids. In some embodiments an
epitope or antigen comprises a portion at least 80%, 85%, 90%, 95%,
96%, 96%, 97%, 98%, 99%, or more identical in sequence to at least
a portion of a naturally occurring polypeptide, wherein the portion
of the naturally occurring polypeptide is at least 10; 20; 30; 40;
50; 100; 200; 500; 1,000; 2,000; 3,000, or more amino acids
long.
[0331] In some embodiments epitopes may be provided as a pool of
peptides, which may be derived from one or more proteins. The
protein(s) may include one or more proteins that are known to be
target(s) of cell-mediated and/or humural immunity in at least some
individuals. In some embodiments a mixture of peptides may contain
at least one epitope capable of binding to MHC class I proteins and
at least one epitope capable of binding to MHC class II proteins.
In some embodiments a mixture of peptides may contain at least one
CTL epitope and at least one Th epitope. A peptide pool may
comprise multiple epitopes that can bind to different MHC alleles.
Peptides in the pool may bind to MHC alleles from individuals of
diverse genetic backgrounds and may be capable of stimulating T
and/or B cells from individuals of diverse genetic backgrounds. The
peptides may be from 8 to 30 amino acids (aa) long, e.g., 9 to 15
aa long, 15 to 25 aa long. In some embodiments the peptides may be
generated by chemical and/or enzymatic partial hydrolysis of longer
protein(s). In some embodiments the peptides comprise a mixture of
overlapping synthetic peptides. Overlapping synthetic peptides
typically represent sequential stretches of amino acids, wherein a
given peptide within the pool overlaps with neighboring peptides by
at least one aa up to n-1 aa, wherein n is the length of the
peptides. The first aa of a given peptide may be offset from the
first aa of its neighboring peptides by from 1 aa up to n-1,
wherein n is the length of the peptides. In some embodiments the
offset is 2, 3, 4, or 5 amino acids. For example, individual
peptides may be 15 aa in length (15 mer) and overlap with their
neighboring peptides by 11 aa (offset=4). Starting at position 1 of
a 30 amino acid polypeptide, such a peptide pool would contain
peptides extending from aa 1-15, aa 5-20, aa 10-25, and aa 15-30.
As another example, peptides may be 20 aa in length, with 10 aa
overlaps between sequential peptides (offset=10). Peptides in a
pool may be, but need not be, the same length. A peptide pool may
include some peptides that fall completely within the sequence of
other peptides. The peptides may cover all or part of the full
length of a polypeptide. In some embodiments a peptide pool
comprises peptides that collectively encompass at least 50%, 60%,
70%, 80%, 90%, 95%, or all 9 peptide sequences of a particular
protein or proteins. The number of different peptides in a peptide
pool may range from 2 up to about 300, up to about 500, up to about
1,000, or more. In some embodiments the number of different
peptides is at least 10, 20, 30, 40, 50, e.g., between 20 and 100,
100 and 200, 200 and 300, 300 and 400, or 400 and 500. The peptides
may be synthesized using standard solid phase peptide synthesis
methods.
[0332] In general, an antigen or epitope that originates from a
particular source may, in various embodiments, be isolated from
such source or may be produced using any appropriate means, e.g.,
using recombinant nucleic acid technology or chemical synthesis, or
combinations thereof. An antigen or epitope may be modified, e.g.,
by conjugation to another molecule or entity (e.g., an adjuvant),
chemical or physical denaturation, etc. In certain embodiments an
antigen or antigen composition comprises or is derived at least in
part from cells or tissues. For example, a tumor or tumor sample
may be removed from a subject and used to isolate or identify one
or more antigens present in the tumor or tumor sample. Such
antigens may be used, e.g., to stimulate immune cells ex vivo, to
generate or select binding agents (e.g., antibodies) that bind to
the antigen(s).
[0333] Antigens may be useful for a variety of purposes. It will be
understood that epitopes derived from a particular antigen may be
used for any such purpose in certain embodiments. For example, an
antigen may be used to identify, generate, test, or use an antibody
or other agent that binds to the antigen or may be conjugated to
another entity, e.g., to a polypeptide or cell. Antigens may be
used in vitro to load or stimulate cells of the immune system. For
example, antigens may be contacted with APCs to cause such APCs to
take up, process, and display epitopes at the cell surface. The
APCs may be administered to a subject or may be used in vitro to
stimulate cells of the adaptive immune system (e.g., naive T and/or
B cells), which may subsequently be administered to a subject.
[0334] Pathogen-derived antigens may be useful in, e.g.,
identifying or detecting pathogens or pathogen-infected cells
(e.g., for purposes of diagnosis of an infection, for purposes of
monitoring subjects who have received treatment for an infection
e.g., to test for recurrence), for purposes of targeting various
agents (e.g., therapeutic agents) to pathogens or pathogen-infected
cells, and/or for purposes of modulating (e.g., directing or
enhancing) an immune response towards pathogens or
pathogen-infected cells. Tumor antigens may be useful in, e.g.,
identifying or detecting tumor cells or tumor-associated cells
(e.g., for purposes of diagnosis, for purposes of monitoring
subjects who have received treatment for a tumor, e.g., to test for
recurrence), for purposes of targeting, e.g., targeting therapeutic
agents or cells, to tumor cells or tumor-associated cells, and/or
for purposes of modulating (e.g., directing or enhancing) an immune
response towards tumor cells or tumor-associated cells.
[0335] Self antigens may be useful in, e.g., identifying or
detecting antibodies or immune system cells that may contribute to
an autoimmune disease (e.g., for purposes of diagnosis of an
autoimmune disease, for purposes of monitoring subjects who have
received treatment for an autoimmune disease, e.g., to test for
recurrence), identifying or detecting self cells or substances
towards which an inappropriate, e.g., harmful or potentially
harmful, immune response is directed in an autoimmune disease
(e.g., for purposes of diagnosis or treatment selection), for
purposes of targeting various agents (e.g., therapeutic agents) to
self cells or substances towards which an inappropriate, e.g.,
harmful or potentially harmful, immune response is directed in an
autoimmune disease, and/or for purposes of modulating (e.g.,
inhibiting) an immune response towards cells or substances towards
which an inappropriate, e.g., harmful or potentially harmful,
immune response is directed in an autoimmune disease.
[0336] Graft-associated antigens may be useful in, e.g.,
identifying or detecting antibodies or immune system cells that may
contribute to graft rejection (e.g., for purposes of diagnosis of
graft rejection, for purposes of monitoring subjects who have
received a graft), identifying or detecting grafted cells towards
which a harmful or potentially harmful immune response is directed
in a subject who has received a graft (e.g., for purposes of
diagnosis or treatment selection), for purposes of targeting
various agents (e.g., therapeutic agents) to grafted cells towards
which a harmful or potentially harmful immune response is directed
in in a subject who has received a graft, and/or for purposes of
modulating (e.g., inhibiting) an immune response towards grafted
cells in a subject who has received a graft.
[0337] In some embodiments an antigen is a target for a targeting
moiety that targets an entity, e.g., a cell, detection agent, or
therapeutic agent, to a pathogen, pathogen-infected cell, tumor
cell, or tumor associated cell. In some embodiments an antigen is a
target for a targeting moiety that targets an entity, e.g., a cell,
detection agent or therapeutic agent, towards a cell or substance
comprising a self antigen or other antigen towards which tolerance
is desired, such as a graft-associated antigen. In some embodiments
an agent conjugated to a living mammalian cell using sortase
comprises a moiety that binds to an antigen, such as an antibody or
antibody fragment.
[0338] In some embodiments a protein comprises an antibody,
antibody fragment, or antibody domain. In some embodiments a
protein comprising an antibody, antibody fragment, or antibody
domain is conjugated to living mammalian cells using sortase. The
cells may be used as a delivery vehicle for the protein and/or the
protein may serve as a targeting moiety to target the cells to a
target to which the antibody, antibody fragment, or antibody domain
binds. In some embodiments a protein is a therapeutic antibody.
Exemplary therapeutic antibodies that are useful in various
embodiments provided herein include, but are not limited to, the
following antibodies (target of the antibody is listed in
parentheses together with exemplary non-limiting therapeutic
indications): Abciximab (glycoprotein IIb/IIIa; cardiovascular
disease), Adalimumab (TNF-.alpha., various auto-immune disorders,
e.g., rheumatoid arthritis), Alemtuzumab (CD52; chronic lymphocytic
leukemia), Basiliximab (IL-2R.alpha. receptor (CD25); transplant
rejection), Bevacizumab (vascular endothelial growth factor A;
various cancers, e.g., colorectal cancer, non-small cell lung
cancer, glioblastoma, kidney cancer; wet age-related macular
degeneration), Blinatumomab (anti-CD3/anti-CD19; various
hematologic malignancies), Brentuximab (CD30; various hematologic
malignancies); Catumaxomab (CD3 and EpCAM; malignant ascites),
Cetuximab (EGF receptor, various cancers, e.g., colorectal cancer,
head and neck cancer), Certolizumab (e.g., Certolizumab pegol) (TNF
alpha; Crohn's disease, rheumatoid arthritis), Daclizumab
(IL-2R.alpha. receptor (CD25); transplant rejection), Eculizumab
(complement protein C5; paroxysmal nocturnal hemoglobinuria),
Efalizumab (CD11a; psoriasis), Elotuzumab (CD1 (also known as
SLAMF7 and as CD319, multiple myeloma); Epratuzumab (CD22;
Non-Hodgkin's lymphoma; lupus; ALL); Gemtuzumab (CD33; acute
myelogenous leukemia (e.g., with calicheamicin)), Ibritumomab
tiuxetan (CD20; Non-Hodgkin lymphoma (e.g., with yttrium-90 or
indium-111)), Infliximab (TNF alpha; various autoimmune disorders,
e.g., rheumatoid arthritis), (Ipilimumab; CTLA-4, melanoma,
prostate cancer), Milatuzumab (CD74; CD74-positive hematologic
malignancies and solid tumors); Muromonab-CD3 (T Cell CD3 receptor;
transplant rejection), Natalizumab (alpha-4 (a4) integrin; multiple
sclerosis, Crohn's disease), Nivolumab (PD-1; cancer, e.g.,
non-small-cell lung cancer, melanoma, and renal-cell cancer);
Omalizumab (IgE; allergy-related asthma); Ofatumumuab (CD20;
Non-Hodgkin lymphoma, chronic lymphocytic leukemia); Obinutuzumab
(CD20; Non-Hodgkin lymphoma, chronic lymphocytic leukemia);
Palivizumab (epitope of RSV F protein; Respiratory Syncytial Virus
infection), Panitumumab (EGF receptor; cancer, e.g., colorectal
cancer), Ranibizumab (vascular endothelial growth factor A; wet
age-related macular degeneration), Rituximab (CD20; Non-Hodgkin
lymphoma), Tositumomab (CD20; Non-Hodgkin lymphoma), Trastuzumab
(ErbB2; breast cancer); Tremelimumab (CTLA-4, melanoma); Veltuzumab
(CD20; Non-Hodgkin lymphoma, chronic lymphocytic leukemia), and any
antigen-binding fragment thereof. In some embodiments an antibody
or other binding agent binds to the same target as any of the
afore-mentioned antibodies. In some embodiments an antibody or
other binding agent competes with any of the afore-mentioned
antibodies for binding to its target. It will be understood that
antigen binding domains of any of the afore-mentioned antibodies or
others described may be used. For example, Fab fragments or single
chain variable fragments (scFv) may be used.
[0339] In some embodiments a binding moiety, e.g., an antibody,
binds to an extracellular domain of a mammalian receptor. In some
embodiments the receptor is overexpressed in a tumor cell as
compared with a normal cell, e.g., a normal cell of the same cell
type, and/or has increased activity in a tumor cell as compared
with a normal cell, e.g., a normal cell of the same cell type. In
some embodiments the receptor is encoded by a gene that is mutated,
is a fusion gene that results from a chromosomal translocation,
and/or is amplified in a tumor cell. In some embodiments the
receptor is a protein kinase, e.g., a tyrosine kinase or a
serine/threonine kinase. In some embodiments the receptor is an
oncogenic protein kinase.
[0340] In some embodiments, a therapeutic monoclonal antibody and a
second agent useful for treating the same disease or comprising a
targeting moiety are conjugated to mammalian cells using sortase.
In some embodiments, the second agent comprises a polypeptide,
peptide, small molecule, or second antibody.
[0341] In some embodiments, a monoclonal antibody and a cytokine,
e.g., an interferon, e.g., interferon alpha, are conjugated to
mammalian cells using sortase. Optionally, the monoclonal antibody
and cytokine are both useful for treating the same disease.
[0342] In some embodiments one or more subunits (e.g., separate
polypeptide chains) of a multisubunit protein (which term is used
interchangeably with multichain protein) is conjugated to mammalian
cells using sortase. In some embodiments, a multisubunit protein is
a receptor (e.g., a cell surface receptor). In some embodiments, a
multisubunit protein is an enzyme. In some embodiments, a
multisubunit protein is a cytokine. In some embodiments, a
multisubunit protein is a channel or transporter. In some
embodiments at least one subunit of a multisubunit protein
comprises a sortase recognition motif, which may be used to
conjugate the subunit to mammalian cells or to conjugate a moiety
to the polypeptide. Various multisubunit polypeptides and methods
of modifying them using sortase are described in WO/2011/133704. In
some embodiments a sortase recognition motif is located in a
flexible loop, which may be cleaved by a protease so as to position
the sortase recognition motif at or near the C-terminus of a
resulting cleavage product. In some embodiments a first subunit of
a multisubunit protein is conjugated to mammalian cells. In some
embodiments one of more additional subunits may subsequently
associate with the first subunit, e.g., to form a complete
multi-subunit protein. In some embodiments such association may
occur in vitro. The one or more additional subunits may be added to
a culture vessel or may be produced by cells in the vessel. In some
embodiments association occurs in vivo after administration of the
cells to a subject. In some embodiments the one or more additional
subunits may be produced by the administered cells, by other cells
in the body of the subject, or may be administered to the subject.
In some embodiments two or more subunits, at least one of which
comprises a sortase recognition motif, assemble to form a
multi-subunit protein before conjugation to mammalian cells. In
some embodiments two or more subunits are covalently linked to each
other directly or via a linker before the protein is conjugated to
mammalian cells. In some embodiments, such linkage facilitates
proper folding of the multi-subunit protein (e.g., accelerates
folding or increases proportion of correctly folded functional
proteins). In some embodiments a subunit of a multi-subunit protein
may be modified using sortase before the subunit or a different
subunit of the protein is conjugated to mammalian cells. For
example, a label may be conjugated to a first subunit using
sortase, and a second subunit may be conjugated to cells. The
various conjugation and/or association steps may occur in any order
in various embodiments.
[0343] In some embodiments a protein comprises a peptide that binds
to a target. In some embodiments the peptide is selected using a
display technology, e.g., phage display, yeast display, ribosome
display, bacterial display, or directed evolution. In some
embodiments the peptide is selected from a peptide library. In some
embodiments a protein may comprise any of a variety of polypeptide
scaffolds known in the art including, e.g., those based on or
incorporating one or more protein folds or domains from, e.g.,
protein Z, fibronectin, ankyrin repeat proteins; cysteine-knot
miniproteins, Armadillo repeat proteins, lipocalins, or stefin A.
In some embodiments a protein comprises an affibody, adnectin,
DARPin, knottin, anticalins, or steffin. The protein, e.g.,
affibody, adnectin, DARPin, knottin, anticalins, or steffin, may be
designed or selected to bind to a target of interest. Such
engineered binding proteins may in some embodiments have a
specificity and/or affinity comparable to or in some embodiments
superior to that of typical antibodies. In some embodiments a
peptide that binds to a target is inserted into a polypeptide
scaffold. See, e.g., Hoffmann, T., et al. Protein Eng Des Sel.,
23(5):403-13, 2010, and references therein, for discussion of
various proteins and polypeptide scaffolds. In some embodiments any
such protein or scaffold is used, e.g., as a binding moiety,
targeting moiety, immunomodulator, or therapeutic agent.
[0344] In some embodiments a binding agent or moiety, e.g., an
antibody, binds to a target antigen, target entity, or binding
partner with a K.sub.D of less than about 10.sup.-6 M, less than
about 10.sup.-7 M, less than about 10.sup.-8 M less than about
10.sup.-9 M, less than about 10.sup.-10 M, less than about
10.sup.-11M, less than about 10.sup.-12M, or less than about
10.sup.-13M. In certain embodiments a binding agent or moiety binds
to a target antigen or target entity or binding partner with a
K.sub.D of between about 10.sup.-6 M and about 10.sup.-13M, e.g.,
between about 10.sup.-6 M and about 10.sup.-7M, between about
10.sup.-7M and about 10.sup.-8M, between about 10.sup.-8M and about
10.sup.-9M, between about 10.sup.-9M and about 10.sup.-10 M,
between about 10.sup.-10 M and about 10.sup.-11 M, or between about
10.sup.-11 M and about 10.sup.-12 M or between about 10.sup.-12 M
and about 10.sup.-13 M. In some embodiments a binding interaction
may have a K.sub.D of between about 10.sup.-16M and about
10.sup.-12M.
[0345] In certain embodiments nucleic acids, e.g., short
interfering RNAs, antisense oligonucleotides, or aptamers, may be
conjugated to mammalian cells using sortase or used in vitro, e.g.,
to promote or inhibit expansion, activation, or differentiation of
cells. In some embodiments nuclieic acid aptamers are of interest,
e.g., as binding moieties, targeting moieties, immunomodulators,
therapeutic agents, ligands. An aptamer comprises an
oligonucleotide that binds specifically and with high affinity to
its target (e.g., a protein target). In some embodiments the
oligonucleotide is single-stranded (although it may in some
embodiments form regions of double-stranded secondary structure
through intramolecular complementarity). An aptamer may be
identified through a selection process using, e.g., systematic
evolution of ligands by exponential enrichment (SELEX), phage
display, or various directed evolution techniques. See, e.g.,
Turek, C. and Gold, L., Science 249: 505-10, 1990; Brody E N and
Gold L J, Biotechnol. J, 74(1):5-13, 2000; L. Cerchia and V. de
Franciscis, Trends Biotechnol., 28: 517-525, 2010; Keefe, A. Nat.
Rev. Drug Discov. 9: 537-550, 2010. Aptamers can be generated using
DNA or RNA backbones, either of which may comprise any of a variety
of modifications such as substitution of ribonucleotides with
2'-amino, 2'-fluoro, or 2'-O-alkyl nucleotides. Aptamers can be
generated against most targets and can inhibit the function of the
proteins to which they bind or may act as agonists to activate a
receptor to which they bind. Aptamers may be, e.g., about 25 to 80
nt long and can be synthesized chemically.
[0346] In some embodiments small molecules may be used, e.g., as
targeting moieties, immunomodulators, detection agents, therapeutic
agents, or as ligands to activate or inhibit a receptor.
[0347] In some embodiments an agent to be conjugated to mammalian
cells comprises an anti-cancer agent (also termed a "chemotherapy
drug"). In certain embodiments cells are conjugated both with an
anti-cancer agent and a targeting moiety, wherein the targeting
moiety targets the cell to a cancer, which in some embodiments is a
cancer of a type that is typically treated with the anti-cancer
agent. In certain embodiments cells conjugated with an anti-cancer
agent and/or with a targeting moiety that targets the cells to a
cancer are administered to a subject who is in need of treatment
for cancer. Any anti-cancer agent may be used in various
embodiments. In some embodiments an anti-cancer agent is a protein,
e.g., a monoclonal antibody. In some embodiments an anti-cancer
agent is an enzyme, e.g., asparaginase. Non-limiting examples of
chemotherapy drugs that may be used include, e.g., alkylating and
alkylating-like agents such as nitrogen mustards (e.g.,
chlorambucil, chlormethine, cyclophosphamide, ifosfamide, and
melphalan), nitrosoureas (e.g., carmustine, fotemustine, lomustine,
streptozocin); platinum agents (e.g., alkylating-like agents such
as carboplatin, cisplatin, oxaliplatin, BBR3464, satraplatin),
busulfan, dacarbazine, procarbazine, temozolomide, thioTEPA,
treosulfan, and uramustine; antimetabolites such as folic acids
(e.g., aminopterin, methotrexate, pemetrexed, raltitrexed); purines
such as cladribine, clofarabine, fludarabine, mercaptopurine,
pentostatin, thioguanine; pyrimidines such as capecitabine,
cytarabine, fluorouracil, floxuridine, gemcitabine; spindle
poisons/mitotic inhibitors such as taxanes (e.g., docetaxel,
paclitaxel), vincas (e.g., vinblastine, vincristine, vindesine, and
vinorelbine), epothilones; cytotoxic/anti-tumor antibiotics such
anthracyclines (e.g., daunorubicin, doxorubicin, epirubicin,
idarubicin, mitoxantrone, pixantrone, and valrubicin), compounds
naturally produced by various species of Streptomyces (e.g.,
actinomycin, bleomycin, mitomycin, plicamycin) and hydroxyurea;
topoisomerase inhibitors such as camptotheca (e.g., camptothecin,
topotecan, irinotecan) and podophyllums (e.g., etoposide,
teniposide); monoclonal antibodies for cancer therapy such as
anti-receptor tyrosine kinases (e.g., cetuximab, panitumumab,
trastuzumab), anti-CD20 (e.g., rituximab and tositumomab), and
others for example alemtuzumab, aevacizumab, gemtuzumab;
photosensitizers such as aminolevulinic acid, methyl
aminolevulinate, porfimer sodium, and verteporfin; tyrosine and/or
serine/threonine kinase inhibitors, e.g., inhibitors of Abl, Kit,
insulin receptor family member(s), VEGF receptor family member(s),
EGF receptor family member(s), PDGF receptor family member(s), FGF
receptor family member(s), mTOR, Raf kinase family, phosphatidyl
inositol (PI) kinases such as PI3 kinase, PI kinase-like kinase
family members, cyclin dependent kinase (CDK) family members,
Aurora kinase family members (e.g., kinase inhibitors that are on
the market or have shown efficacy in at least one phase III trial
in tumors, such as cediranib, crizotinib, dasatinib, erlotinib,
gefitinib, imatinib, lapatinib, nilotinib, sorafenib, sunitinib,
vandetanib), growth factor receptor antagonists, and others such as
retinoids (e.g., alitretinoin and tretinoin), altretamine,
amsacrine, anagrelide, arsenic trioxide, asparaginase (e.g.,
pegasparagase), bexarotene, bortezomib, denileukin diftitox,
estramustine, ixabepilone, masoprocol, mitotane, and testolactone,
Hsp90 inhibitors, proteasome inhibitors (e.g., bortezomib),
angiogenesis inhibitors, e.g., anti-vascular endothelial growth
factor agents such as bevacizumab (Avastin) or VEGF receptor
antagonists or soluble VEGF receptor domain (e.g., VEGF-Trap),
matrix metalloproteinase inhibitors, various pro-apoptotic agents
(e.g., apoptosis inducers), Ras inhibitors, anti-inflammatory
agents, cancer vaccines, or other immunomodulating therapies, RNAi
agents targeted to oncogenes, etc. It will be understood that the
preceding classification is non-limiting. A number of anti-tumor
agents have multiple activities or mechanisms of action and could
be classified in multiple categories or classes or have additional
mechanisms of action or targets.
[0348] In some embodiments an agent to be conjugated to mammalian
cells comprises an anti-microbial agent. As used herein,
anti-microbial agents include compounds that inhibit proliferation
or activity of, weaken, destroy, or kill bacteria, viruses, fungi,
parasites (e.g., protozoa, helminths (whether or not microscopic)
compounds that inhibit invasion of cells by viruses, bacteria, or
parasites; compounds that inhibit one or more steps of a viral,
bacterial, fungal, or parasite life cycle. In certain embodiments
cells are conjugated with an anti-microbial agent suitable for use
against a bacteria, virus, fungi, or parasite and with a targeting
moiety, wherein the targeting moiety targets the cell to the
bacteria, virus, fungi, or parasite or targets the cell to a cell
infected by the bacteria, virus, fungi, or parasite.
[0349] One of skill in the art will be aware of or can readily
obtain the sequences of proteins described herein or other proteins
of interest. Naturally occurring sequences, e.g., genomic, mRNA,
and polypeptide sequences, from a wide variety of species,
including human, are known in the art and are available in publicly
accessible databases such as those available at the National Center
for Biotechnology Information (www.ncbi.nih.gov) or Universal
Protein Resource (www.uniprot.org). Databases include, e.g.,
GenBank, RefSeq, Gene, UniProtKB/SwissProt, UniProtKB/Trembl, and
the like. In general, sequences, e.g., nucleic acid (e.g., mRNA)
and polypeptide sequences, in the NCBI Reference Sequence database
may be used as reference sequences. It will be appreciated that
multiple alleles of a gene may exist among individuals of the same
species. For example, differences in one or more nucleotides (e.g.,
up to about 1%, 2%, 3-5% of the nucleotides) of the nucleic acids
encoding a particular protein may exist among individuals of a
given species. Due to the degeneracy of the genetic code, such
variations often do not alter the encoded amino acid sequence,
although DNA polymorphisms that lead to changes in the sequence of
the encoded proteins can exist. Examples of polymorphic variants
can be found in, e.g., the Single Nucleotide Polymorphism Database
(dbSNP), available at the NCBI website at
www.ncbi.nlm.nih.gov/projects/SNP/. (Sherry S T, et al. (2001).
"dbSNP: the NCBI database of genetic variation". Nucleic Acids Res.
29 (1): 308-311; Kitts A, and Sherry S, (2009). The single
nucleotide polymorphism database (dbSNP) of nucleotide sequence
variation in The NCBI Handbook [Internet]. McEntyre J, Ostell J,
editors. Bethesda (Md.): National Center for Biotechnology
Information (US); 2002
(www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=handbook&part=ch5).
Multiple isoforms of certain proteins may exist, e.g., as a result
of alternative RNA splicing or editing. In general, where aspects
of this disclosure pertain to a gene or gene product, embodiments
pertaining to allelic variants or isoforms are encompassed unless
indicated otherwise. Certain embodiments may be directed to
particular sequence(s), e.g., particular allele(s) or isoform(s).
It will be understood that a polypeptide may be encoded by any of
numerous different nucleic acid sequences due to the degeneracy of
the genetic code. If a polypeptide is produced recombinantly, a
nucleic acid sequence encoding the polypeptide may be selected or
codon optimized for expression in a particular species, if desired.
It should be understood that wherever reference is made herein to a
protein or polypeptide, e.g., a naturally occurring protein or
polypeptide, the invention provides embodiments in which a variant
or fragment, e.g., a functional variant or fragment, may be used.
(See discussion of variants and fragments above). For example, it
will be understood that an enzyme conjugated to mammalian cells for
purposes of supplementing or replacing an enzyme that is lacking or
present in insufficient amounts need not be identical in sequence
to a naturally occurring enzyme that is lacking or insufficient, so
long as it provides the appropriate catalytic activity.
[0350] In some embodiments a protein or other moiety conjugated to
mammalian cells is PEGylated. In some embodiments PEGylation is may
be accomplished using sortase, e.g., before the protein or other
moiety is conjugated to mammalian cells. For example, a protein may
be PEGylated at its N-terminus and then conjugated to mammalian
cells via its C-terminus. In some embodiments sortase is used to
conjugate a moiety comprising PEG to mammalian cells.
[0351] Sortagged cells, e.g., sortagged mammalian cells, described
herein have a number of uses. Some of these uses are described
herein, but the invention is not limited to uses described herein.
In some embodiments sortagged mammalian cells may be used in cell
therapy. As used herein, the terms "cell therapy", "cell-based
therapy", or "cellular therapy" are used interchangeably to refer
to administration of eukaryotic cells, e.g., mammalian cells, to a
subject for therapeutic purposes. In some embodiments cell therapy
comprises cell-based immunotherapy, which refers to administration
of cells to a subject in order to modulate or augment the subject's
immune system or immune response for therapeutic purposes.
Cell-based immunotherapy encompasses administering immune system
cells to a subject, wherein the administered cells or their
descendants, through their own effector mechanisms and/or through
interactions with cells or substances of the subject's immune
system, may provide a therapeutic benefit to the subject. It will
be understood that where the present disclosure refers to the
effects of administered cells, such effects encompass the effects
of the administered cells and their descendants that are generated
in vivo. In some embodiments cell therapy is administered for
treatment of cancer, infections, autoimmune diseases, or enzyme
deficiencies. In certain embodiments administered cells may
originate from the individual to whom they are administered
(autologous), may originate from different genetically identical
individual(s) of the same species (isogeneic), may originate from
different non-genetically identical individual(s) of the same
species (allogeneic), or may originate from individual(s) of a
different species. In certain embodiments allogeneic cells may
originate from an individual who is immunocompatible with the
subject to whom the cells are administered.
[0352] In some embodiments living mammalian cells modified by
sortase-mediated conjugation of an agent thereto are used as
delivery vehicles for the agent. For example, living mammalian
cells that have a protein conjugated to their surface may serve as
delivery vehicles for the protein. Such cells may be administered
to a subject suffering from a deficiency of the protein or who may
benefit from increased levels of the protein. In some embodiments
the cells are administered to the circulatory system, e.g., by
infusion. In some embodiments the cells are administered
intravenously. For example, hematopoietic cells, e.g., RBCs, WBCs,
platelets, may be administered to the circulatory system. In some
embodiments the cells are administered to the lymphatic system,
e.g., by infusion. In some embodiments the protein is one that is
normally present in the blood, e.g., a protein produced by the
liver or by hematopoietic cells or endothelial cells. In some
embodiments the protein is an enzyme. The enzyme may be
catalytically active or may become catalytically active after
administration. In some embodiments the enzyme may act on a
substrate in the blood. In some embodiments at least a portion of
the protein attached to sortagged may be released from the cells in
vivo. Release may occur via cleavage that, in some embodiments,
also activates the protein. For example, a protein may be attached
as an inactive enzyme precursor (zymogen). In some embodiments
sortase-modified cells are administered locally, e.g., to a tissue
or organ in which a protein or a substrate of the protein is
normally produced or active. Examples of various diseases
associated with deficiency of various proteins, e.g., enzymes, are
provided above.
[0353] In certain embodiments mammalian cells conjugated with an
anti-microbial agent and/or with a targeting moiety that targets
the cells to a microbe or parasite may be administered to a subject
who is at risk of infection or is infected by the microbe or
parasite, e.g., a subject who has been recently exposed to the
microbe or parasite, has been determined to harbor detectable
levels of the microbe or parasite (e.g., in the blood), or is
clinically ill with an infection caused by the microbe or parasite.
The cells may be administered to the circulatory system or locally
at or near a site of infection.
[0354] In certain embodiments mammalian cells conjugated with an
agent that inhibits a toxic or harmful substance maybe administered
to a subject who has been exposed to the toxic or harmful substance
and is at risk of toxicity or damage from the substance, a subject
who exhibits symptoms of exposure, or a subject who is at risk of
exposure. For example, a subject who is infected by or has been
exposed to a toxin-producing pathogen that may be treated with
cells that have been conjugated with an agent that inhibits the
toxin.
[0355] In some aspects, sortase is used to modify mammalian cells
that may be used in a cell based immunotherapy. For example, in
some embodiments one or more epitopes or antigens (e.g., epitopes
or antigens of pathogens or tumors) may be conjugated to mammalian
cells using sortase. In some embodiments the mammalian cells are
hematopoietic cells, e.g., RBCs. In some embodiments the mammalian
cells are fibroblasts. In some embodiments, the sortase-modified
cells may be administered to a subject in order to induce or
enhance an immune response against the antigen or cells comprising
the antigen(s), e.g., pathogens, pathogen-infected cells, tumor
cells, toxic substances, etc. In some embodiments, the
sortase-modified cells may be used ex vivo, e.g., to stimulate or
activate other cells, e.g., immune system cells such as T cells,
antigen-presenting cells (e.g., dendritic cells, macrophages), B
cells, NK cells, or precursors of any such cells. In some aspects,
the present disclosure provides methods of producing or modifying a
cellular artificial APC. For example, in some embodiments, a cell
is sortagged with one or more moieties (e.g., antigens,
TCR-engaging molecules, costimulatory molecules) that allows it to
serve as an aAPC or improves its ability to serve as an aAPC, e.g.,
to provide costimulation. In some embodiments cells that have been
stimulated ex vivo by contact with a sortase-modified cell are
administered to a subject, e.g., to induce or enhance an immune
response directed to a pathogen, pathogen-infected cells, tumor,
toxic substance, etc.
[0356] In some embodiments, sortase-modified mammalian immune
system cells are used in adoptive immunotherapy. "Adoptive
immunotherapy", also called "adoptive cell transfer" (ACT) refers
to administration (transfer) of immune system cells to a subject,
e.g., for therapeutic purposes. In some embodiments administered
immune system cells exert an immune response against a tumor,
pathogen, or pathogen-infected cells. For example, cytotoxic T
cells (e.g., CD8+ T cells), NK cells, or phagocytes (e.g.,
neutrophils, macrophages) may exert direct cytotoxic effects on
target cells. In some embodiments target cells may be tumor cells,
tumor-associated cells, pathogens, pathogen-infected cells, or
other unwanted cells. In some embodiments target cells may be
immune system cells that exert an abnormal or damaging effect on
self cells or tissues. For example, target cells may be
autoreactive T cell clones, plasma cells that produce
autoantibodies, etc.
[0357] In certain embodiments immune system cells (e.g., cytotoxic
T cells, NK cells, CD4+ T cells, DCs, neutrophils, macrophages) are
sortagged with a targeting moiety that targets the cells to a tumor
cell, tumor-associated cell, pathogen, pathogen-infected cell, or
other unwanted target. The targeting moiety may be any moiety that
binds to the target. The targeting moiety may, for example,
comprise an antibody, antibody fragment, engineered polypeptide,
aptamer, or ligand (e.g., small molecule ligand). In some
embodiments the targeting moiety binds to a tumor antigen. The
targeting moiety targets the cell to tumor cells that express the
tumor antigen. Binding agents (e.g., monoclonal antibodies) that
specifically bind to a variety of different tumor antigens are
known in the art, and additional binding agents can be identified
using methods known in the art. For example, trastuzumab binds to
Her2/Neu and may be used as a targeting moiety to target tumors
that express Her2/Neu (e.g., certain breast cancers). Rituximab
binds to CD20 and may be used as a targeting moiety to target
malignancies that express CD20 (e.g., non-Hodgkin's lymphoma (e.g.,
follicular lymphoma), diffuse large B cell lymphoma, mantle cell
lymphoma). In certain embodiments the sortagged cells mount an
immune response or promote an endogenous immune response against
tumor cells, tumor-associated cells, pathogens, pathogen-infected
cells, or other unwanted cells or substances to which they are
targeted. The immune response may comprise cell-mediated
cytotoxicity, antibody production, release of cytokines or other
agents that damage the target cells, etc. In some embodiments the
administered cells may have been exposed in vitro to one or more
antigens expressed by a tumor cell, tumor-associated cell,
pathogen, or pathogen-infected cell. The cells may be exposed prior
to or after sortagging. In some embodiments the cells may have been
exposed ex vivo to APCs that present an epitope of an antigen
expressed by a tumor cell, tumor-associated cell, pathogen, or
pathogen-infected cell. In some embodiments the cells are exposed
in vitro to agents that stimulate the cells to proliferate,
differentiate, or become activated, such as anti-CD3 antibodies,
co-stimulators, etc. In some embodiments, targeting APCs, e.g.,
DCs, to a tumor, pathogen, pathogen-infected cell, or other target
results in increased presentation of target-associated antigens by
the APCs, which may enhance an immune response mounted by
endogenous or administered immune system cells. In some
embodiments, targeting CD4+ T cells to a tumor, pathogen,
pathogen-infected cell, or other target results in increased
provision of "helper" functions by such cells, which may enhance an
immune response mounted by endogenous or administered immune system
cells that receive such help.
[0358] In some embodiments mammalian cells are sortagged with an
agent comprising a substance that is capable of exerting toxic
effects on a target cell, e.g., a tumor cell, tumor-associated
cell, pathogen, or pathogen-infected cell. In some embodiments the
cells are administered to a subject and deliver the toxic substance
to a target such as a tumor. In some embodiments the toxic
substance comprises a pro-apoptotic agent, a cytolytic agent, a
cytotoxic drug, or a toxin. In some embodiments the target cells
are relatively susceptible to the toxic effect of the toxic
substance as compared with the sortagged cells. For example, a
sortagged cell may on average remain viable when sortagged with the
toxic substance and able to deliver the toxic substance to a target
cell in an amount that is lethal to the target cell. The target
cells may differ from the sortagged cells in one or more ways that
renders the target cells more susceptible to effects of the toxic
substance. Differences in tumor cell metabolism, proliferation
rate, or oncogene expression, as compared with most non-tumor cells
may be exploited to select agents that are selectively toxic to
tumor cells. In some embodiments the sortagged cells may either
naturally or as a result of engineering lack a receptor for the
toxic substance or may lack a molecular target or functional
biological pathway by which the toxic substance acts or otherwise
be rendered relatively less susceptible to effects of the toxic
substance.
[0359] In some embodiments mammalian cells, e.g., immune system
cells, may be sortagged with an agent comprising a cytolytic
domain. For example, cells may be sortagged with a perforin,
granzyme, granulysin or biologically active domain thereof. Such a
cytolytic domain may increase the cytotoxic activity of the cells
(e.g., if the cells are cytotoxic cells) or may confer cytotoxic
activity on cells that would otherwise lack it. In some embodiments
a cytolytic domain comprises or is derived from a cytolytic agent
that is naturally found in a subject, e.g., one that is normally
used by immune system cells of the subject to lyse cells. In some
embodiments a cytolytic domain comprises or is derived from a
cytolytic agent that is not naturally found in a subject. For
example, a variety of cytolytic toxins, e.g., cytolysins produced
by various microbes, are known (see, e.g., discussion below). An
agent may further comprise a targeting moiety that targets the
cells to target cells to be lysed, e.g., tumor cells, infected
cells, pathogens. In some embodiments a targeting moiety is
separately conjugated to the cells.
[0360] In some embodiments mammalian cells, e.g., immune system
cells, may be sortagged with a pro-apoptotic agent. In some
embodiments a pro-apoptotic agent is an agent comprising a domain
that promotes apoptosis (pro-apoptotic domain). In some embodiments
a pro-apoptotic agent is an agent that can initiate or enhance an
apoptotic pathway if introduced into or activated in a cell. As
known in the art, apoptosis is a process of programmed cell death
that occurs in multicellular organisms, e.g., mammals. A
pro-apoptotic domain may be any domain that can deliver an
extracellular signal that initiates or enhances an apoptotic
pathway. In some embodiments an apoptotic pathway leads to
activation of caspases, e.g., initiator caspases, which in turn
activate effector caspases. Effector caspases proteolytically
degrade a variety of intracellular proteins to carry out the cell
death program. Pro-apoptotic domains include, e.g., ligands that
bind to TNF alpha receptors (e.g., TNF-R1) or Fas in mammalian
cells. In some embodiments a pro-apoptotic domain may comprise any
moiety that can activate TNF-R1 or Fas. In some embodiments a
pro-apoptotic domain comprises FasL, Trail, Tweak, Lymphotoxin, TNF
alpha, or a biologically active domain thereof. A pro-apoptotic
domain or other pro-apoptotic agent may increase the cytotoxic
activity of the cells (e.g., if the cells are cytotoxic cells) or
may confer cytotoxic activity on cells that would otherwise lack
it. In some embodiments a pro-apoptotic domain or other
pro-apoptotic agent comprises or is derived from a protein that is
naturally found in a subject, e.g., one that is normally used by
immune system cells of the subject or during development or other
physiological processes that involve apoptosis. In some embodiments
a pro-apoptotic domain or other pro-apoptotic agent comprises or is
derived from a substance that is not naturally found in a subject.
In some embodiments a pro-apoptotic agent comprises a TRAIL
receptor agonist. TRAIL receptors include TRAIL-R1 (also known as
death receptor 4 (DR4) and TRAIL-R2 (also known as death receptor 5
(DR5)). TRAIL receptor agonists include the natural ligand TRAIL
and other agents that bind to a TRAIL receptor and mimic the effect
of TRAIL. In some embodiments the TRAIL receptor agonist is a
monoclonal antibody that binds to TRAIL-R1 and/or TRAIL-R2, such as
Mapatumumab (human anti-DR4 mAb), Tigatuzumab (humanized anti-DR5
mAb), Lexatumumab (human anti-DR5 mAb), Conatumumab (human anti-DR5
mAb), Apomab (human anti-DR5 mAb). In some embodiments TRAIL or a
TRAIL receptor agonist is useful in treatment of breast, colon,
lung, pancreatic, prostate, renal carcinoma, thyroid carcinoma,
glioma, multiple myeloma or leukemia. In some embodiments a
pro-apoptotic agent induces apoptosis by inhibiting an
anti-apoptotic protein such as Bc1-2, Bc1-xL, and Bcl-w or by
activating a pro-apoptotic protein such as BAX, BID, BAK, or BAD
inside a cell. For example, a pro-apoptotic agent may be a member
of the BH3-only family of proteins or a biologically active domain
thereof. BH3-only proteins inhibit antiapoptotic members of the
Bcl-2 family. Examples of such proteins include NOXA or a
biologically active fragment thereof (e.g., the BH3 domain or the
mitochondrial targeting domain). In some embodiments the
pro-apoptotic agent comprises a small molecule BH3 mimetic such as
ABT-737, ABT-263 (navitoclax) or ABT-199 (Vandenberg C J and Cory,
S., Blood. 2013; 121(12):2285-8 and references therein). In some
embodiments, a pro-apoptotic domain is useful to treat a cancer
that has increased expression or activity of an anti-apoptotic
protein as compared with non-cancer cells. In some embodiments, a
pro-apoptotic domain is useful to treat a cancer that has become
resistant to cytotoxic chemotherapy or to inhibit emergence of such
resistance. A pro-apoptotic agent may further comprise a targeting
moiety that targets the cells to target cells whose apoptosis is
desired e.g., tumor cells, infected cells. In some embodiments a
targeting moiety is separately conjugated to the cells.
[0361] In certain embodiments mammalian cells, e.g., immune system
cells, are sortagged with an immunomodulator. In some embodiments
the immunomodulator promotes survival, proliferation,
differentiation and/or one or more activities of at least some of
the administered cells. In some embodiments the immunomodulator
comprises a stimulatory cytokine, costimulatory molecule (e.g.,
OX40, OX40L, CD137L), or adjuvant (e.g., a CD40 ligand, anti-CD40
antibody, TLR ligand). In certain embodiments immune system cells
are sortagged with a targeting moiety and an immunomodulator. A
targeting moiety and an immunomodulator may be provided as
individual agents or may be part of a bifunctional agent.
[0362] In some embodiments administered immune system cells may
modulate immune system cells that are co-administered to the
subject or already present in the subject. For example,
administered immune system cells may stimulate or inhibit
proliferation, activation, differentiation, migration, and/or
maturation of immune system cells present in the subject.
Stimulation or inhibition may result at least in part from
secretion of cytokine(s) by the transferred cells and/or from
cell-cell interactions (e.g., display of costimulatory molecules or
inhibitory molecules such as CD28/CTLA-4 family members (e.g.,
CTLA-4 or PD-1 ligand) by the transferred cells). In some
embodiments a moiety conjugated to immune system cells may comprise
a costimulator. Such cells may provide costimulation at a tumor
site or site of infection, which may, for example, enhance the
ability of tumor-specific or pathogen-specific T cells to eliminate
tumor cells or infected cells. Whether transferred cells stimulate
or inhibit the immune system of the subject may depend on a variety
of factors, such as the properties of the particular cell type
transferred and/or the identity and/or amount of the agent
conjugated to the transferred cells. Such factors may be selected
according to the desired effect of the cells. Transferred CD4+ T
cells may provide "help" to endogenous or co-administered cytotoxic
cells, which may expand and/or augment the ability of cytotoxic
cells to eliminate target cells. Transferred Treg cells may
suppress immune responses of endogenous immune system cells that
may otherwise exert unwanted or deleterious activity against the
subject's own cells or tissues or against transplanted cells or
tissues. Such suppression may be useful to treat autoimmune
diseases or reduce the likelihood of rejection of a transplant.
[0363] In some embodiments immune system cells comprise a
polyclonal population, in that the population comprises multiple
subpopulations of cells that express TCRs or BCRs that are specific
for a variety of different targets, antigens, or epitopes. For
example, a polyclonal population may comprise T and/or B cells that
collectively express at least 10.sup.3, 10.sup.4, 10.sup.5, or more
distinct TCRs or BCRs. A polyclonal population of lymphocytes may
be obtained, e.g., from peripheral blood. In some embodiments
immune system cells comprise a monoclonal population of T or B
cells in that cells in the population carry TCRs or BCRs that are
specific for a particular epitope, which may be an epitope present
in or on a target such as a tumor cell, tumor-associated cell,
pathogen, pathogen-infected cell, or other unwanted cell. In some
embodiments two or more monoclonal populations may be combined,
e.g., populations having TCRs or BCRs specific for different
epitopes of a target antigen or different antigens of a target
entity.
[0364] In some embodiments autologous or allogeneic T cells with
anti-tumor activity are obtained, optionally expanded and/or
activated in vitro, and sortagged, (e.g., with a targeting moiety,
biologically active moiety, or both). In some embodiments the
sortagged T cells are introduced into a subject in need of
treatment for cancer. In some embodiments T cells comprise
autologous tumor-infiltrating lymphocytes (TILs). Autologous TILs
may be obtained using methods known in the art from a tumor
following biopsy or removal of the tumor from the subject. In some
embodiments allogeneic T cells are a T cell line, e.g., the NK-92
cell line or a derivative thereof.
[0365] In some embodiments autologous or allogeneic NK cells with
anti-tumor activity are obtained, optionally expanded and/or
activated in vitro, and sortagged, (e.g., with a targeting moiety,
biologically active moiety, or both). In some embodiments the
sortagged NK cells are introduced into a subject in need of
treatment for cancer. In some embodiments allogeneic NK cells are
an NK cell line, e.g., the NK-92 cell line or a derivative
thereof.
[0366] In some embodiments immune system cells may be contacted in
vitro with one or more epitopes, e.g., in order to activate cells
that recognize such epitope(s). In some embodiments immune system
cells may be contacted in vitro with microbes or parasites, tumor
cells, tumor tissue, pathogen-infected cells, cells of a tumor cell
line, material (e.g., proteins, RNA, membrane fraction, lysate)
derived from such cells or tissue, or partly purified or synthetic
antigens or epitopes (e.g., a peptide pool). In some embodiments
immune system cells (e.g., T cells) that bind to or proliferate in
response to such cells or substances may be isolated. The immune
system cells (e.g., T cells) may be sortagged, e.g., with a
targeting moiety that targets them to a tumor or pathogen, and
administered to a subject who is in need of treatment of a tumor or
who is infected or at risk of infection by the pathogen. In some
embodiments the cells or substances used to stimulate or isolate
the immune system cells are derived from a particular patient's
tumor or comprise TA(s) or TA epitopes found in a particular
patient's tumor (or typically found in tumors of that type), and
the sortagged immune system cells are administered to the
patient.
[0367] In some embodiments a method may comprise isolating or
determining the identity of one or more antigens or epitopes
expressed by tumor cells or tumor-associated cells obtained or
originating from a subject and conjugating a targeting moiety that
binds to at least one of the antigens or epitopes to mammalian
cells in vitro. The antigens or epitopes may be identified or
isolated using any of a variety of methods used in the art. RNA
(e.g., mRNA) or proteins from tumors, tumor cells, or tumor tissue
samples can be analyzed using standard methods for RNA or protein
detection and quantification. For example, proteins may be analyzed
using immunological methods such as immunohistochemistry or ELISA.
In some embodiments the cells comprise immune system cells, e.g.,
cytotoxic cells, e.g., cytotoxic T cells or NK cells. In some
embodiments a cytotoxic cell releases proteins (e.g., cytolytic
proteins) that induce lysis of a target cell. In some embodiments a
cytotoxic cell is able to induce apoptosis in a target cell. In
some embodiments the cells comprise T helper cells. The method may
further comprise administering the cells to the subject. In some
embodiments a method may comprise isolating or determining the
identity of one or more antigens expressed by tumor cells or
tumor-associated cells obtained or originating from a subject, and
conjugating a targeting moiety that binds to at least one of the
antigen(s) to immune system cells ex vivo. The method may further
comprise administering the cells to the subject.
[0368] In some embodiments, a tumor sample is analyzed to identify
one or more TAs expressed by the tumor, to which therapeutic cells
are to be targeted. The tumor sample may be from a tumor removed at
surgery, from a biopsy, blood sample (e.g., cells of a hematologic
malignancy or circulating tumor cells from a solid tumor). A panel
of antibodies or other binding agents may be used to identify cell
surface TAs. In some embodiments, a patient who has been treated
for a tumor or is suspected of having a tumor or tumor recurrence
may be monitored by performing periodic blood tests to detect a
soluble tumor antigen or circulating tumor cells. If test results
show an increase or abnormally high level of the soluble TA or
presence of circulating tumor cells, a therapeutic cell composition
of the present invention, comprising cells targeted to a TA, may be
administered. For example, a patient who has been or is being
treated for a tumor that expresses CA-125 may be monitored to
detect increased blood levels of CA-125. If such increased levels
are detected, a therapeutic cell composition comprising cells
targeted to CA-125 and/or targeted to a different TA expressed by
the tumor may be administered.
[0369] In some embodiments, cells are conjugated to a first
targeting moiety that binds to a first tumor antigen and a second
targeting moiety that binds to a second tumor antigen, wherein the
first and second tumor antigens are expressed by cells of the same
tumor (tumor cells and/or tumor associated cells). The first and
second TAs may be expressed by different cell populations in the
tumor or may be expressed on at least some of the same cells of the
tumor. The targeting moieties may be part of the same agent, e.g.,
a bispecific antibody, or may be two separate agents. In some
embodiments, two cell populations are admininstered to a subject in
need of treatment for a tumor: a first cell population targeted to
a first TA and a second cell population targeted to a second TA.
For example, as noted above, certain tumors express CA125 and
mesothelin. Cells to be administered to a subject in need of
treatment for a tumor may be conjugated to a first targeting moiety
that binds to CA125 and a second targeting moiety that binds to
mesothelin or a first cell population targeted to mesothelin and a
second cell population targeted to CA125 may be administered. In
some embodiments, administration of a cell targeted to two or more
different tumor antigens may have an additive or greater than
additive effect. In some embodiments, administration of two cell
populations, each targeted to a different tumor antigen, may have
an additive or greater than additive effect.
[0370] In some embodiments, cells, e.g., immune system cells, are
sortagged with an agent comprising a targeting moiety that targets
the cells to circulating tumor cells (CTCs). CTCs are cells from a
tumor (e.g., a solid tumor) that intravasate into the circulation
(vascular system and/or lymphatic system). CTCs may extravasate at
sites to which they are carried by the circulation, where they may
survive and form metastases. CTCs can interact with receptors on
endothelial cell walls in a way that resembles leukocyte
extravasation in inflammation and lymphocyte homing. CTCs from many
types of primary tumors express sialylated carbohydrate ligands
similar to those of leukocytes, which mediate interactions with
selectins on the endothelium. In some embodiments a selectin is
used as a targeting moiety to target cells, e.g., immune system
cells, to circulating tumor cells (CTCs). In some embodiments the
cells are sortagged both with a selectin and with one or more
additional agents. In some embodiments the one or more additional
agents comprise a cytotoxic moiety, a detectable moiety, a second
targeting moiety (e.g., a targeting moiety that binds to a TA
expressed by the tumor), or any combination thereof. In some
embodiments the cytotoxic moiety comprises a pro-apoptotic agent,
e.g., a pro-apoptotic protein such as TRAIL or a biologically
active portion thereof or a TRAIL receptor agonist.
[0371] In some embodiments cells sortagged with a detectable moiety
and targeted to CTCs may be useful to detect the presence of CTCs
in a sample (e.g., a blood sample) obtained from a subject or in
vivo. The subject may be suspected of having cancer or may have
been treated for cancer. Without wishing to be bound by any theory,
the fact that a cell may be sortagged with numerous individual
molecules of a detectable moiety may facilitate detection of CTCs,
e.g., by making detection more reliable (e.g., fewer false positive
and/or false negative results) and/or by permitting detection of
smaller numbers of CTCs than with various other methods.
[0372] Cancer stem cells (CSCs) are cancer cells found within
tumors or hematological cancers that possess characteristics
analogous to characteristics associated with normal stem cells.
CSCs have the capacity to initiate tumors, self-renew, and
differentiate into phenotypically diverse cancer cells. CSCs are
often relatively resistant to chemotherapy drugs and radiation and
are proposed to persist and cause relapse and metastasis by giving
rise to new tumors. CSCs may express a variety of cell surface
markers at levels that differentiate them from non-CSC cancer cells
and/or most normal cells. For example, CD133, CD44, and EpCAM have
been identified as CSC markers in a variety of epithelial cancers.
In some embodiments a binding moiety, e.g., an antibody, that binds
to a CSC marker is used as a targeting moiety to target cells,
e.g., immune system cells, to CSCs. In some embodiments the cells
are sortagged both with a CSC marker and with one or more
additional agents. In some embodiments the one or more additional
agents comprise a cytotoxic moiety, a detectable moiety, a second
targeting moiety (e.g., a targeting moiety that binds to a TA
expressed by non-CSC tumor cells or tumor-associated cells), or any
combination thereof. In some embodiments the cytotoxic moiety
comprises a pro-apoptotic agent, e.g., a pro-apoptotic protein such
as TRAIL or a biologically active portion thereof or a TRAIL
receptor agonist.
[0373] In some embodiments APCs are contacted in vitro with tumor
cells, tumor tissue, pathogen-infected cells, material (e.g.,
proteins, RNA, membrane fraction, lysate) derived from such cells
or tissue, or partly purified or synthetic antigens or epitopes
(e.g., a peptide pool) and are then used to stimulate lymphocytes
in vitro. In some embodiments the stimulated lymphocytes may be
sortagged (e.g., with a targeting moiety that targets them to a
tumor, with a chemotherapeutic agent potentially active against the
tumor, and/or with an immunomodulator) and may be administered to a
subject who is in need of treatment of a tumor or is at risk of
developing a tumor or of tumor recurrence. In some embodiments the
stimulated lymphocytes may be sortagged (e.g., with a targeting
moiety that targets them to a pathogen or pathogen-infected cell,
with an antimicrobial agent potentially active against the
pathogen, and/or with an immunomodulator) and may be administered
to a subject who is in need of treatment of an infection or is at
risk of infection by the pathogen.
[0374] Protocols for T cell activation and/or expansion may
include, e.g., culturing the cells in medium containing appropriate
cytokines such as interleukin-2 (IL-2) and/or appropriate
co-stimulatory molecules. In some embodiments an expansion protocol
using IL-2 and CD3 ligation via an anti-CD3 antibody may be used
(so-called "rapid expansion method", e.g., as described in Dudley M
E, et al., Generation of tumor-infiltrating lymphocyte cultures for
use in adoptive transfer therapy for melanoma patients. J
Immunother 2003, 26:332-342). The cytokine(s) may be provided as
isolated proteins or by co-culture with cells that secrete them
either naturally or as a result of genetic manipulation. In some
embodiments cells are cultured in the presence of anti-CD3
antibodies and anti-CD28 antibodies. In some embodiments, the
antibodies are attached to beads (e.g., paramagnetic beads) or
another support, such as the interior sides and bottom of a cell
culture vessel. In some embodiments, a mixture of anti-CD3 and
anti-CD28 antibodies is attached to the beads (coimmobilization)
(see, e.g., Levine, B., et al., Tumaini, B., et al. (both cited
above), and various other references cited herein. In some
embodiments, cells are co-cultured with PBMCs, which comprise cells
that provide one or more molecules that promote activation and/or
expansion. In some embodiments cells are cultured with PBMC and
anti-CD28 antibodies (e.g., attached to a support such as beads).
In some embodiments the PBMCs are immunocompatible with the T
cells. In some embodiments both the T cells and PBMCs are derived
from a subject to whom the T cells are to be introduced. In some
embodiments a ratio of beads to cells between 100:1 and 1:100 is
used. In some embodiments, a ratio of beads to cells between 10:1
and 1:10, between 5:1 and 1; 5, between 3:1 and 1:3, or about 1:1
is used.
[0375] In some embodiments lymphocytes are activated in vitro by
contacting them with APCs. In some embodiments artificial APCs may
be used. A variety of cellular aAPCs are known in the art. For
example, K562 cells (available from the ATCC, Manassas, Va.) can
serve as artificial APC. Such cells have been engineered to express
a variety of co-stimulatory molecules and used for ex vivo
expansion of polyclonal and antigen-specific cytotoxic T
lymphocytes, natural killer cells, and antigen-experienced
tumor-infiltrating lymphocytes (Maus M V, et al. Nat Biotechnol
2002, 20:143-148; Suhoski M M, et al. Mol Ther 2007, 15:981-988;
Fujisaki, H., Cancer Res 2009; 69: 4010-4017; Ye Q, et al., J
Transl Med 2011, 9:131; Butler, M O, et al., Clin Cancer Res Mar.
15, 2007 13; 1857). Cellular aAPC (e.g., a cellular aAPC that has
been genetically engineered and/or has been or is to be sortagged)
may be irradiated (e.g., with gamma radiation, e.g., about 100 Gy
in certain embodiments) or otherwise rendered unable to
proliferate. In some embodiments noncellular aAPCs may be used.
Noncellular aAPCs may be particles that have antigen presenting
molecules (APMs) attached thereto. Such particles may be contacted
with antigen or antigen fragment (e.g., peptides) to allow them to
serve as aAPCs. In some embodiments, noncellular APCs may be
particles such as liposomes, beads (e.g., paramagnetic beads),
particles comprised at least in part of organic polymers, quantum
dots, etc. Such particles may have any of a variety of different
molecules attached to their cell surface, e.g., any of the
molecules discussed above in reference to cellular aAPC.
Noncellular aAPCs may be generated as described in Oelke M, et al.,
Ex vivo induction and expansion of antigen-specific cytotoxic T
cells by HLA-Ig-coated artificial antigen-presenting cells. Nat
Med. 2003, 9:619-625; Webb, T, et. al., J Immunol Methods, 2009,
346(1-2): 38-44; Oelke M. and Schneck, J P, Immunol Res (2010);
47:248-256; East, J E, J Vis Exp. Artificial antigen presenting
cell (aAPC) mediated activation and expansion of natural killer T
cells. 2012 Dec. 29; (70). pii: 4333. doi: 10.3791/4333. In some
embodiments APMs may be conjugated to a support, e.g., beads (e.g.,
magnetic beads), inner surface of a vessel such as a well or plate,
etc. APCs may be loaded with antigen prior to or after being
conjugated to a support. Cells to which the antigen is to be
presented are contacted with the support to permit antigen
presentation to occur. In some embodiments an APM may be conjugated
or fused to an IgG domain or other moiety that may serve as a
linker to attach the APM to a support.
[0376] In some embodiments lymphocytes, e.g., T cells, are
stimulated in vitro by co-culturing them with APCs, e.g., DCs or
aAPCs. In some embodiments the APCs display at least one epitope
that stimulates proliferation and/or effector activity of T cells
that bind to it. In some embodiments an agent comprising one or
more epitopes is conjugated to the APCs, e.g., using sortase. In
some embodiments an agent comprising one or more epitopes is
non-covalently bound to a cell surface molecule or complex
expressed by APCs. In some embodiments the cell-surface molecule or
complex is Dec-205, a C type lectin such as DC-SIGN, or MHC class
II (MHCII) protein. In some embodiments the cells have been
sortagged or the cells and/or descendants thereof are subsequently
sortagged according to methods described herein. In some
embodiments sortagged mammalian cells are administered to a
subject.
[0377] In some embodiments a moiety conjugated to immune system
cells using sortase may inhibit or overcome mechanisms that may
exist in a subject that may otherwise limit the therapeutic
efficacy of cellular immunotherapy or may limit the effectiveness
of the subject's endogenous immune system. In the case of therapy
for cancer, such mechanisms may include immune evasion mediated by
the secretion of immunosuppressive substances such as transforming
growth factor .beta. (TGF.beta.) in the microenvironment of a tumor
and/or mediated by the accumulation of regulatory T cells, both of
which can, for example, dampen the in vivo activation, expansion,
and tumor homing of transferred tumor-reactive CD8+ T cells. In the
case of therapy for diseases caused by pathogens, such mechanisms
may include any of various immunoevasive or immunosuppressive
agents produced or encoded or induced by pathogens. A moiety
conjugated to cells may inhibit the effect of an immunoevasive or
immunsuppressive substance by, for example, binding to the
substance, acting as an antagonist or competitor at a receptor for
the substance, or antagonizing a pathway activated by the
substance. In some embodiments a moiety conjugated to cells may
inhibit development or activity of Tregs that would otherwise
dampen an immune response against a tumor, pathogen, or
pathogen-infected cell.
[0378] "Immune checkpoint pathways" or "immune checkpoints" are
naturally existing inhibitory pathways of the immune system that
play important roles in maintaining self-tolerance and modulating
the duration and level of effector output (e.g., in the case of T
cells, the levels of cytokine production, proliferation or target
killing potential) of physiological immune responses in order to
minimize damage to the tissues of the individual mounting the
immune response. Such pathways may, for example, downmodulate T
cell activity or enhance regulatory T cell immunosuppressive
activity. Examples of immune checkpoint pathways include, e.g., the
PD-1 pathway and the CTLA-4 pathway, discussed further below.
Tumors frequently co-opt certain immune-checkpoint pathways as a
major mechanism of immune resistance, e.g., against T cells that
are specific for tumor antigens. Certain aspects of the invention
utilize sortase-modified cells to inhibit immune checkpoint
mechanisms. In some aspects, the invention provides mammalian cells
conjugated using sortase to a moiety comprising an immune
checkpoint modulator. In some embodiments the immune checkpoint
modulator is an immune checkpoint inhibitor. "Immune checkpoint
inhibitor" refers to any agent that inhibits (suppresses, reduces
activity of) an immune checkpoint pathway. In some embodiments the
immune checkpoint modulator is an immune checkpoint activator.
"Immune checkpoint activator" refers to any agent that activates
(stimulates, increases activity of) an immune checkpoint pathway.
In some embodiments the cells are non-genetically modified cells.
In some embodiments the cells are derived from a subject in need of
treatment for cancer or an immunocompatible donor. In some
embodiments the cells comprise PBMCs or RBCs. In some embodiments
the cells comprise lymphocytes. "Immune checkpoint protein" refers
to those proteins that are components of immune checkpoint
pathways, and include membrane-bound, soluble (e.g., secreted), and
intracellular proteins. Many immune checkpoint pathways are
initiated by interactions between membrane-bound receptors and
soluble or membrane-bound ligands. In some embodiments an immune
checkpoint inhibitor is an agent that binds to receptor or ligand
that is a component of an immune checkpoint pathway. Binding of the
agent to the receptor or ligand blocks the ligand-receptor
interaction, thus inhibiting the immune checkpoint pathway. For
example, in some embodiments a moiety inhibits an interaction
between PD-1 and a PD-1 ligand, e.g., by binding to either PD-1 or
PD-1 ligand.
[0379] In some embodiments an agent comprises a modulator of the
PD-1 pathway. PD-1 is an inhibitory surface receptor expressed by a
variety of cells, including activated T cells, B cells, natural
killer T cells, monocytes, and dendritic cells (DC). PD-1 has two
naturally occurring ligands, programmed cell death ligand 1 (PD-L1)
and programmed cell death ligand 2 (PD-L2), also called B7-H1 and
B7-DC, respectively. The term "PD-L" refers to either or both PD-L1
and PD-L2. Where the term "PD-L" is used herein, certain
embodiments pertain to PD-L1, certain embodiments pertain to PD-L2,
and certain embodiments pertain to both PD-L1 and PD-L2. The term
"PD-1 pathway" refers to the biological processes that occur in a
cell, e.g., an immune system cell, upon binding of PD-L to PD-1
expressed by the cell. Binding of PD-L to PD-1 on immune system
cells, e.g., helper or cytotoxic T cells, typically has inhibitory
effects on their proliferation and/or activity, particularly in the
context of stimulation by antigen, and may contribute to T cell
exhaustion. However, the PD-1 pathway can promote the development
and activity of T regulatory cells, which may suppress the activity
of other immune system cells (e.g., helper and/or cytotoxic T
cells) that would otherwise mount an immune response, e.g., against
a tumor, infected cell, pathogen, or a self antigen (e.g., in a
subject with an autoimmune disease). Tumors can use the PD-1
pathway to inhibit immune system cells that may otherwise mount an
immune response against the tumor. Certain tumors express PD-L1,
and its expression has been correlated with tumor aggressiveness
and inversely correlated with survival of patients, possibly
because natural antitumor immunity against such tumors is
inhibited. The PD-1 pathway has also been shown to impair immune
function in various infections such as influenza virus infection,
HIV infection, HCV infection, and Mycobacterial infection. In some
embodiments, inhibiting the PD-1 pathway is of use in treatment of
conditions in which the PD-1 pathway inhibits the immune response
of administered immune system cells (or their descendants) or
endogenous immune system cells against a tumor, pathogen, or
infected cell and/or in which activity of the PD-1 pathway is
abnormally or inappropriately high or in which there is an
abnormally or inappropriately high number and/or activity of Tregs.
In some embodiments, activating the PD-1 pathway is of use in
treatment of conditions in which activity of the PD-1 pathway is
abnormally or inappropriately low, conditions in which there is an
abnormally low number and/or activity of Tregs, and/or conditions
in which increased number or activity of Tregs may be beneficial,
e.g., in subjects at risk of or suffering from an autoimmune
diseases, GVHD, and/or transplant rejection. A number of cells or
level of activity may be abnormally or inappropriately high or low
in absolute terms or relative to the number or activity of immune
systems cells of one or more other types or subtype, e.g., Th1,
Th2, Th17, cytotoxic T cells, helper T cells. In some embodiments,
a modulator of the PD-1 pathway is of use to restore an appropriate
balance between Tregs and other immune system cells.
[0380] In some embodiments a modulator of the PD-1 pathway is an
inhibitor of the PD-1 pathway. An inhibitor of the PD-1 pathway
reduces activity of the PD-1 pathway as compared to the activity of
the pathway in the absence of the inhibitor. It will be understood
that the effect of an inhibitor may be observable only in the
presence of a ligand that would (in the absence of the inhibitor)
bind to PD-1 and activate it. An inhibitor of the PD-1 pathway may
be referred to as a PD-1 antagonist. In some embodiments a PD-1
antagonist binds to PD-1 or PD-L and blocks the interaction between
PD-1 and PD-L. In some embodiments a PD-1 antagonist comprises a
nucleic acid (e.g., a nucleic acid aptamer), protein, peptide, or
small molecule that binds to PD-1, PD-L1, or PD-L2, wherein if the
PD-1 antagonist binds to PD-1, it does not significantly stimulate
PD-1 signaling. In some embodiments, an anti-PD-1, anti-PD-L1, or
anti-PD-L2 antibody may be used as a PD-1 antagonist. Nivolumab (a
fully human IgG4 monoclonal antibody), CT-011 (a humanized IgG1
monoclonal antibody), and lambrolizumab (also known as MK-3475, a
humanized IgG4 monoclonal antibody) are examples of anti-PD-1
antibodies. BMS-936559 (a fully human IgG4 monoclonal antibody),
MPDL3280A (human monoclonal, Genentech), and MEDI4736 (Medimmune)
are anti-PD-L1 antibodies.
[0381] In some embodiments, a PD-1 antagonist comprises at least a
portion of the extracellular domain of PD-1 or a variant or
fragment thereof that binds to PD-L. If a cell is genetically
engineered to express a variant or fragment of PD-1, a variant or
fragment that substantially lacks the ability to inhibit cells that
express it may be used. For example, the variant or fragment may
have a mutation or at least partial deletion of its intracellular
signaling domain. The variant or fragment may comprise the
transmembrane domain and, optionally, at least a portion of the
intracellular domain or may comprise a transmembrane domain from a
different protein or a synthetic transmembrane domain to cause it
to remain attached to the cell surface rather than secreted as a
soluble protein. The extracellular domain of PD-1 or a variant or
fragment thereof may serve as a targeting moiety to target cells,
e.g., T cells or NK cells, to a tumor comprising cells that express
PD-L (e.g., PD-L1). By binding to PD-L at the surface of tumor
cells or tumor-associated cells or infected cells that express
PD-L, PD-1 extracellular domain may alternately or additionally
serve to block the effect of PD-L on immune system cells that
express PD-1. Thus, the immune suppressive effects of PD-L, e.g.,
PD-L1, expressed in tumors or infected cells, may be reduced. A
variety of PD-1 antagonists, e.g., antibodies that bind to PD-1,
PD-L1, or PD-L2 are described in U.S. Pat. Pub. No. 20040213795,
20110195068, 20120039906, 20120114649, 20130095098, 20130108651,
20130109843, 20130237580, and 20130291136, among others.
[0382] In some embodiments a modulator of the PD-1 pathway
increases activity of the PD-1 pathway, e.g., by binding to and
activating PD-1. A modulator of the PD-1 pathway that binds to and
activates PD-1 may be referred to as a PD-1 agonist. A PD-1 agonist
may comprise a biologically active fragment or variant of the
extracellular domain of PD-L1 or PD-L2 or may mimic the effect
induced by binding of PD-L1 or PD-L2 to PD-1. Cells that are
sortagged with a PD-1 agonist may be useful in conditions in which
it is desired to inhibit the immune response.
[0383] An agent that binds to PD-1 or PD-L may serve as a targeting
moiety. For example, an agent that binds to PD-L may target cells
that express or are sortagged with the agent to cells that express
PD-1. The agent may also serve as a PD-1 agonist or antagonist in
various embodiments. An agent that binds to PD-1 may target cells
that express or are sortagged with the agent to cells that express
PD-L. The agent may also serve as a PD-1 agonist or antagonist in
various embodiments.
[0384] In some embodiments an agent comprises a modulator of a T
cell co-inhibitory receptor, such as the anti-inflammatory receptor
cytotoxic T lymphocyte antigen-4 (CTLA-4). CTLA-4 is an important
negative regulator of T cell activation. In some embodiments a
modulator of CTLA-4 inhibits CTLA-4 activity and may be referred to
as a "CTLA-1 antagonist". Inhibiting negative regulation mediated
by CTLA-4 has been shown to promote stimulation of adaptive
immunity and potentiation of T cell activation. Binding of a CTLA-4
ligand (e.g., B7-1 or B7-2) to CTLA-4 can reduce T cell
proliferation and functional activity and promote tolerance, which
may reduce the ability of administered immune system cells and/or
endogenous immune system cells of a subject to mount an immune
response against a tumor, pathogen, or infected cells. Accordingly,
in some embodiments a CTLA-4 antagonist is useful to limit or
prevent negative regulation by CTLA-4. In some embodiments, a
CTLA-4 antagonist comprises an agent that binds to CTLA-4 and
blocks CTLA-4 ligands from binding to CTLA-4. For example,
anti-CTLA-4 antibodies, such as ipilimumab or tremelimumab, may be
used. In some embodiments a CTLA-4 antagonist comprises an antibody
that binds to a CTLA-4 ligand. In addition to CTLA-4, other T cell
co-inhibitory receptors including B7 family members B7-H3, B7-H4, T
cell immunoglobulin and mucin domain-containing protein 3 (Tim-3),
and lymphocyte activation gene-3 (LAG-3), interact with their
cognate ligands on various cells types, including APCs, regulatory
T cells (Tregs), and nonhematopoietic cells, resulting in reduced T
cell proliferation and functional activity. BTLA is an inhibitory
receptor on T cells. Its ligand is herpesvirus entry mediator
(HVEM), which is expressed on certain tumor cell types such as
melanoma and on tumor-associated endothelial cells.
[0385] In some embodiments, an agent inhibits production or
immunosuppressive effect of adenosine. Such an agent may be
referred to as an anti-adenosine agent. Adenosine is produced in
the extracellular compartment by two ectonucleotidases: CD39, which
hydrolyzes ATP and ADP into AMP, and CD73, which converts AMP into
adenosine. Adenosine may also be released from dying cells, e.g.,
dying cells in a tumor. CD73 is expressed on tumor cells and host
immune system cells, including Tregs and myeloid-derived suppressor
cells, and is known to inhibit T-cell proliferation and reduce
cytokine production and cytotoxicity of activated T-cells via A2a
receptor (A2aR) subtype activation, protecting the tumour from
immune-mediated destruction. Adenosine A2a receptor (A2aR), the
ligand of which is adenosine (which may be released from dying
cells in a tumor), inhibits T cell responses in part by driving
CD4+ T cells to develop into Treg cells. In some embodiments an
anti-adenosine agent is an inhibitor of CD73 or CD39. In some
embodiments an anti-adenosine agent comprises an antibody or other
binding moiety that binds to CD73 or CD39. Small molecules that are
selective inhibitors of CD73 include, e.g., adenosine
5'-(.alpha.,.beta.-methylene) diphosphate (APCP) and ZM241365. In
some embodiments an anti-adenosine agent comprises an antibody or
other binding moiety that binds to an A2aR and inhibits its
activity by, e.g., blocking binding of adenosine.
[0386] In some embodiments, an agent that inhibits the biological
activity of any T cell co-inhibitory receptor may be conjugated to
cells to reduce loss of T cell proliferation and/or preserve
functional activity and/or inhibit any immune checkpoint pathway.
For example, antibodies or other binding moieties that bind to
B7-H3, BTLA, A2aR, B7-H4, Tim-3, or LAG-3 may be conjugated to
cells to be administered to a subject, e.g., a subject with cancer.
In some embodiments an antagonist of B7-H3, BTLA, A2aR, B7-H4,
Tim-3, or LAG-3, such as antibodies or other binding moieties that
bind to B7-H3, BTLA, A2aR, B7-H4, Tim-3, or LAG-3 or to a ligand of
B7-H3, BTLA, A2aR, B7-H4, Tim-3, or LAG-3 may be conjugated to
cells to be administered to a subject, e.g., a subject with cancer.
For example, the antibody or other binding moiety may bind to
galectin-9 (ligand of Tim-3), HVEM, or adenosine. In some
embodiments cells are sortagged with two or more different immune
checkpoint inhibitors. The inhibitors may inhibit different immune
checkpoint pathways. For example, cells may be sortagged with at
least two inhibitors selected from PD-1 antagonists, CTLA-4
antagonists, B7-H3 antagonists, BTLA antagonists, A2aR antagonists
or other anti-adenosine agents, B7-H4 antagonists, Tim-3
antagonists, or LAG-3 antagonists.
[0387] Cells may be sortagged with a mixture of agents, e.g., any
of the afore-mentioned immune checkpoint inhibitor, or separate
aliquots of cells may each be sortagged with a single immune
checkpoint inhibitor and administered in combination to a subject,
e.g., a subject in need of treatment for cancer. In some
embodiments cells are sortagged with a first immune checkpoint
inhibitor and then with a second immune checkpoint inhibitor or are
sortagged with two or more immune checkpoint inhibitors at the same
time. In some embodiments cells are sortagged with a first immune
checkpoint inhibitor and administered in combination with a second
immune checkpoint inhibitor. For example, cells may be sortagged
with a PD-1 antagonist and administered in combination with a
CTLA-4 antagonist not attached to cells. In some embodiments cells
that have been sortagged with an immune checkpoint inhibitor are
administered in combination with a targeted cancer therapy such as
a protein kinase inhibitor, e.g., a VEGF receptor inhibitor, RAF
inhibitor, ALK inhibitor, EGF receptor inhibitor, ERB-B1 inhibitor,
ERB-B2 inhibitor, FGF receptor inhibitor, PDGF receptor inhibitor,
etc.
[0388] Without wishing to be bound by any theory, and without
limiting the invention in any way, the use of cells, e.g., RBCs or
T cells, that are conjugated with a targeting moiety (e.g., an
antibody) that binds to a tumor antigen, or that express an antigen
receptor (e.g., a chimeric antigen receptor) that binds to a tumor
antigen, to deliver one or multiple immune checkpoint inhibitors,
may more effectively direct the immune checkpoint inhibitors to the
tumor, increase local activity, and/or reduce unwanted systemic
exposure and associated side effects as compared, for example, with
administering the immune checkpoint inhibitors not attached to
cells. For example, by combining an antibody directed to a tumor
associated antigen with one or more immune checkpoint inhibitors,
the checkpoint inhibitors may be more effectively directed to the
tumor, increasing local activity, and reducing unwanted systemic
exposure and associated side effects. Again without wishing to be
bound by any theory, and without limiting the invention in any way,
using cells, e.g., RBCs, T cells, NK cells, or others, that have
been sortagged with agents such as bi-specific agents comprising
one arm directed to a tumor associated antigen and another arm
directed to CD3, to deliver such agents, the half-life of such
agents may be extended, thereby improving their efficacy without
increasing the amount or frequency of dosing and/or may allow a
decrease in the amount or frequency of dosing without dimishing
efficacy. In some embodiments the cells used to deliver a
bispecific agent may be modified by sortase to carry one or more
antibodies to tumor associated antigens.
[0389] In some embodiments a subject who is to be treated with
cells that have been sortagged with an immune checkpoint inhibitor
has a cancer that has been tested for expression of an immune
checkpoint protein, e.g., a receptor or ligand that is a component
of an immune checkpoint pathway. In some embodiments tumor cells,
non-transformed cells in the tumor stroma, or both, express the
immune checkpoint protein, or overexpress the immune checkpoint
protein, as compared to its expression in normal cells outside the
tumor. In some embodiments, a tumor or tumor sample (e.g., a biopsy
or surgical sample) is analyzed to identify one or more immune
checkpoint proteins that are expressed or overexpressed relative to
normal cells by the tumor cells, non-transformed cells in the tumor
stroma, or both. In some embodiments cells that are sortagged with
an inhibitor of a particular immune checkpoint pathway that is
identified as being active in a tumor are administered to the
subject. In some embodiments cells that are sortagged with an
antagonist of a particular immune checkpoint protein that is
identified as being expressed or overexpressed in a tumor are
administered to the subject. In some embodiments cells that are
sortagged with an antagonist of a receptor or ligand of a
particular immune checkpoint protein that is identified as being
expressed or overexpressed in a tumor may be administered to the
subject. In some embodiments a method comprises identifying an
immune checkpoint pathway that is active or an immune checkpoint
protein that is expressed in a tumor or tumor sample and
administering cells that are sortagged with an agent that inhibits
the immune checkpoint pathway or immune checkpoint protein to a
subject in need of treatment for the tumor. Expression of the
immune checkpoint protein or activity of the immune checkpoint
pathway may be measured using any method known in the art. In some
embodiments the level of mRNA encoding an immune checkpoint protein
may be measured. In some embodiments the level of the protein may
be measured, e.g., using an immunological assay such as an ELISA
assay, immunohistochemistry, or other suitable methods.
[0390] In some embodiments a modulator of CTLA-4 activates CTLA-4
and may be referred to as a "CTLA-4 agonist". In some embodiments a
CTLA-4 agonist is useful to reduce T cell proliferation and
functional activity and/or promote tolerance, e.g., in a subject at
risk or suffering from an autoimmune disease, GVHD, or transplant
rejection.
[0391] In some embodiments, an agent comprises an activator of
CD137 (CD137 agonist). CD137 (also known as 4-1BB), a member of the
tumor necrosis factor (TNF) receptor superfamily and is a T cell
costimulator molecule. Certain anti-CD137 monoclonal antibodies
activate CD137 and are able to activate CD8+ T cells, causing them
to produce interferon (IFN)-.gamma., and induce cytolytic markers.
BMS-663513 (Urelumab), a humanized anti-CD137 antibody, is an
example of a CD-137 agonist.
[0392] In some embodiments, cells are modified to comprise a
targeting moiety and a second agent at their surface. Either the
targeting moiety, second agent, or both, may be conjugated to the
cells using sortase. In some embodiments, the cells are genetically
engineered to express the targeting moiety and are conjugated with
the second agent using sortase. In some embodiments, the cells are
genetically engineered to express the second agent and are
conjugated with the targeting moiety using sortase. In some
embodiments, the targeting moiety, the second agent, or both, are
conjugated via a sortase recognition sequence to an endogenous,
non-genetically engineered polypeptide expressed by the cells. In
some embodiments the cells are not genetically modified to express
a polypeptide comprising an extracellular domain capable of serving
as a sortase substrate or nucleophile in a sortase-catalyzed
reaction. In some embodiments, the cells comprise a chimeric
antigen receptor that comprises an antigen binding moiety that
targets the cells to particular target cells, e.g., tumor cells,
infected cells, or other undesired cells. The second agent can, in
general, comprise any of the various agents described herein. In
some embodiments the second agent comprises a targeting moiety. In
some embodiments the second agent comprises at least a biologically
active portion of a cytokine or chemokine. In some embodiments the
second agent has a biological activity that enhances the biological
activity of the administered cells, e.g., against target cells
(e.g., tumor cells or infected cells) in the body of a subject. In
some embodiments the biological activity is anti-tumor activity,
costimulatory activity towards endogenous immune system cells or
towards administered immune system cells or their descendants, or
inhibitory activity towards endogenous immune system cells that
contribute to an undesired immune response such as in autoimmune
disease or transplant rejection. In some embodiments the second
agent inhibits a biological activity that would otherwise inhibit
or suppress activity of the administered cells or of endogenous
cells, e.g., against target cells (e.g., tumor cells or infected
cells) in the body of a subject.
[0393] In some embodiments a targeting moiety binds to a molecule
expressed in tumor vasculature (a "tumor vasculature marker"). In
some embodiments the tumor vasculature marker is overexpressed in
tumor vasculature as compared with normal vasculature. In some
embodiments the tumor vasculature marker is expressed at the
luminal surface of endothelial cells in tumor vasculature. In some
embodiments the molecule expressed on tumor vasculature is PD-1,
VEGFR1, VEGFR2, tumor endothelial marker 1 (TEM1; also known as
endosialin or CD248), or integrin .alpha.v.beta.3. In some
embodiments the targeting moiety comprises one or more binding
moieties, e.g., antibodies, that bind to PD-1, VEGFR1, VEGFR2,
TEM1, or integrin .alpha.v.beta.3.
[0394] In some embodiments cells to be used in cancer therapy may
be sortagged with an agent comprising a targeting moiety that binds
to a tumor vasculature marker and administered to a subject. In
some embodiments the cells are immune system cells, which in some
embodiments may be sortagged with a second agent, wherein the
second agent has anti-tumor activity, such as an immune checkpoint
inhibitor. In some embodiments the cells are red blood cells that
are sortagged with an agent that has anti-tumor activity, such as
an immune checkpoint inhibitor. In some embodiments the cells are
CAR cells.
[0395] In some aspects the disclosure provides a bispecific agent
comprising (a) a first binding moiety that binds to a molecule on a
target cell; (b) a second binding moiety that binds to a molecule
on an immune system cell, e.g., a T cell, NK cell, or professional
phagocyte (e.g., a monocyte, macrophage, or dendritic cell); and
(c) a sortase recognition motif. In some embodiments the SRM is
appropriately positioned to permit the agent to participate in a
sortase-catalyzed reaction. In some aspects the disclosure provides
a mammalian cell, e.g., a human cell, sortagged with such a
bispecific agent. In some embodiments the target cell is a tumor
cell, tumor-associated cell, pathogen-infected cell, or pathogen.
In some embodiments the target cell is a tumor cell or
tumor-associated cell, and the first binding moiety binds to a
tumor antigen. In some embodiments the second moiety binds to a
molecule on the surface of a T cell, e.g., CD3 (e.g., CD3 delta
chain, CD3 epsilon chain, CD3 gamma chain, CD3 zeta chain, or other
components of the TCR-CD3 complex, e.g., TCR alpha or TCR beta
chains) or CD28. In some embodiments the second binding moiety
binds to a molecule on the surface of an NK cell, dendritic cell,
or macrophage, e.g., an Fc receptor, e.g., an Fc.gamma.receptor,
e.g., Fc.gamma.RI (CD64) or Fc.gamma.RIIIA (CD16a). Other molecules
expressed at the surface of T cell, NK cells, and/or phagocytes are
known to those of ordinary skill in the art.
[0396] In some embodiments the bispecific agent links the sortagged
cell to the immune system cell and to the target cell. The immune
system cell is thereby linked to the target cell via the sortagged
cell. In some embodiments, linking the immune system cell to the
target cell via the sortagged cell causes one or more effector
functions of the immune system cell (e.g., cytotoxicity) to be
directed towards the target cell. Alternatively, or additionally,
in certain embodiments in which the sortagged cell is an immune
system cell, one or more effector functions of the sortagged cell
may be directed towards the target cell. In some embodiments, the
individual or combined effect of attack by the immune system cell,
the sortagged cell, or both, results in death of the target cell.
In some embodiments two or more immune system cells are linked to
the sortagged cell via the bispecific agents attached to the
sortagged cell. An individual sortagged cell may thus tether
multiple immune system cells to a target cell. It should be noted
that multiple target cells may be located in close proximity to
each other. For example, tumor cells are often located close to
each other and/or close to tumor-associated cells in a tumor. An
immune system cell that is linked to a target cell, e.g., via a
sortagged cell, may attack other target cells in close proximity to
the target cell to which it is linked.
[0397] In some embodiments, binding of the second binding moiety to
a molecule on a T cell, NK cell, or phagocyte stimulates such cell.
For example, binding of the second binding moiety to CD3 or CD28 on
a T cell may stimulate the T cell to proliferate, become activated,
secrete cytokines, phagocytose, or exert cytotoxic effects on a
target cell by, e.g., releasing cytotoxins and/or inducing
apoptosis. Binding of the second binding moiety to an Fc receptor
on an NK cell, dendritic cell, or macrophage may stimulate such
cell. For example, an NK cell may be stimulated to exert cytotoxic
effects on a target cell; a DC may be stimulated to secrete
cytokines and/or provide cell-cell interactions that stimulate
other immune system cells; a macrophage may be stimulated to
phagocytose a target cell. In some embodiments the sortagged cell
stimulates the immune system cell to which it is bound, or vice
versa. For example, either cell may secrete one or more cytokines
or provide stimulation via cell-cell contact (e.g., one cell may
express a ligand for a costimulatory receptor on the other cell).
In some embodiments the sortagged cell is also sortagged with an
agent that stimulates the immune system cell or inhibits
immunosuppression of the immune system cell. In some embodiments,
activation is at least in part dependent on the presence of cells
expressing the target antigen to which the first binding moiety
binds. For example, stimulation may occur when the T cell, NK cell,
or phagocyte and the sortagged cell are localized at the site of a
target cell. In some embodiments the sortagged cell is sortagged
with two or more distinct bispecific agents, wherein the second
binding moieties of the distinct bispecific agent differ in regard
to the cell type or molecule to which they bind. An individual
sortagged cell may thus bind to cells of two or more different cell
types, e.g., a T cell and a dendritic cell, or a T cell and a
macrophage, in addition to a target cell.
[0398] In some aspects the disclosure provides a trivalent agent
comprising (a) a first binding moiety that binds to a molecule on a
target cell; (b) a second binding moiety that binds to a molecule
on an immune system cell, e.g., a T cell, NK cell, or phagocyte
(e.g., a monocyte, macrophage, or dendritic cell); (c) a second
binding moiety that binds to a molecule on an immune system cell,
e.g., a T cell, NK cell, or professional phagocyte (e.g., a
monocyte, macrophage, or dendritic cell); and (d) a sortase
recognition motif. In some embodiments the SRM is appropriately
positioned to permit the agent to participate in a
sortase-catalyzed reaction. In some aspects the disclosure provides
a mammalian cell, e.g., a human cell, sortagged with such a
trivalent agent. The first binding moiety may be any binding moiety
that binds to a molecule on a target cell. The second and third
binding moieties may be the same or different and may be any
binding moieties that bind to a T cell, NK cell, or professional
phagocyte. The trivalent agent may link the sortagged cell to two
other cells and to a target cell. The sortagged cell is linked to
the target cell via the first binding moiety, and the two cells to
which the second and third binding moieties bind are linked to the
target cell via the sortagged cell.
[0399] In general, mammalian cell(s) to be sortagged with the
bispecific or trivalent agent may be of any cell type or types in
various embodiments. In some embodiments the cells comprise PBMCs,
lymphocytes, NK cells, APCs, red blood cells, or platelets. The
cells may be a purified population or may be a mixed population. In
some embodiments at least 5%, 10%, 20%, 30%, 40%, or 50% of the
cells are CD4+ T cells. In some embodiments no more than about 1%,
5%, 10%, 15%, 20%, 25%, 30%, 40%, or 50% of the cells are cytotoxic
cells. In some embodiments the cells are not activated ex vivo to
become effector cells. In some embodiments the cells are not
genetically engineered. In some embodiments the cells are
genetically engineered, e.g., they may be CAR cells, may express a
recombinant gene product comprising a TCR, cytokine, cytokine
receptor, costimulator, or costimulator receptor, or agent that
inhibits the effect or production of immunosuppressive substances
produced by tumors, pathogens, or Tregs. In certain embodiments the
sortagged mammalian cells, in addition to being sortagged with the
bispecific or trivalent agent, are also sortagged with one or more
agents that inhibit the effect or production of immunosuppressive
substances produced by tumors, pathogens, or Tregs. Suitable agents
are discussed herein.
[0400] In some embodiments mammalian cells sortagged with the
bispecific agent or trivalent agent are administered to a subject,
and the second binding moiety (and, optionally, the third binding
moiety, if present) binds to an immune system cell in vivo. In
general, an immune system cell to which the second or third binding
moiety binds may be an endogenous immune system cell of a subject
or may be an exogenous cell that has been administered to a
subject. If the immune system cell is an exogenous cell, the cell
may in some embodiments have been sortagged and/or genetically
engineered as described herein. For example, the cell may be a CAR
cell, may produce a recombinant gene product comprising a cytokine,
cytokine receptor, costimulator, costimulator receptor, adhesion
molecule, etc.
[0401] In general, the binding moieties may be of any type, e.g.,
proteins (e.g., antibodies, antibody fragments), nucleic acid
aptamers, small molecules, etc. In some embodiments, the first
binding moiety, second moiety, or both, comprises an antibody,
antibody fragment, scFv, single domain antibody, or any other
moiety that comprises an antigen binding domain. In some
embodiments the bispecific agent is a bispecific antibody. In some
embodiments the bispecific antibody comprises two scFv, wherein a
first say binds to a molecule on a target cell, and the second scFv
binds to a molecule on the surface of an immune system cell, e.g.,
a T cell, NK cell, or professional phagocyte. In some embodiments
the bispecific antibody comprises two single domain antibodies
(sdAb), wherein a first sdAb binds to a molecule on a target cell,
and the second sdAb binds to a molecule on the surface of an immune
system cell, e.g., a T cell, NK cell, or professional phagocyte. In
some embodiments the bispecific antibody comprises an scFv and an
sdAb, wherein either the scFv or the sdAb binds to the target cell
and the other binds to the immune system cell. In some embodiments
the two scFv, two sdAbs, or scFv and sdAb are attached to each
other using click chemistry to form the bispecific agent, and the
bispecific agent is attached to the cell using sortase. In some
embodiments, sortase is used to install click chemistry handles on
the two scFv, two sdAbs, or saFv and sdAb, and the click chemistry
handles are allowed to react, thus generating the bispecific agent.
In some embodiments the two scFv, two sdAbs, or scFv and sdAb are
produced as a fusion protein to form the bispecific agent, and the
bispecific agent is attached to the cell using sortase. In some
embodiments a single polypeptide chain comprising two VH and two VL
regions, optionally separated by spacer regions, is produced, and
the bispecific agent is attached to the cell using sortase. A
sortase recognition motif may, be located at or near a C-terminus
of the agent, which may be used to attach the agent to the cell. In
certain embodiments the bispecific agent is a bispecific antibody,
in some embodiments a bispecific antibody comprising two sdAbs,
which may in some embodiments be attached to each other using click
chemistry, and then attached to the cell using sortase. In some
embodiments, one arm of the bispecific agent recognizes an
activating molecule on a T cell, such as CD3 on T cells, and the
other arm recognizes an antigen on a target cell, such as a tumor
cell. In some embodiments a bispecific agent comprises a first scFv
that binds to CD3 epsilon chain and a second scFv that binds to
CD19, EpCam, CD33, or any other tumor antigen. In some embodiments,
for example, the bispecific antibody blinatumomab
(anti-CD3/anti-CD19), MT103 (anti-CD3/anti-EpCam), or AMG330 may be
modified to comprise a sortase recognition motif and used to sortag
mammalian cells, e.g., human immune system cells. In some
embodiments an scFv from any of the afore-mentioned bispecific
antibodies may be combined with a scFv or other binding moiety that
binds to any tumor antigen. In some embodiments a humanized
anti-CD3 scFv may be used, e.g., a humanized OKT3 scFv (see, e.g.,
Woodle E S, et al. J Immunol 1992; 148:2756-63; Kipriyanov S M, et
al. Protein Eng 1997; 10:445-53). In some embodiments a fully human
anti-CD3 scFv may be used. In some embodiments a bispecific agent
comprises two immune checkpoint inhibitors, optionally that inhibit
different immune checkpoint proteins. In some embodiments a
bispecific agent comprises an immune checkpoint inhibitor and a
targeting moiety. In some embodiments the targeting moiety targets
the cell to tumor cells or tumor vasculature.
[0402] In some embodiments cells are sortagged with or administered
in combination with an agent that enhances immune system cell
infiltration, e.g., T cell infiltration, into tumors. In some
embodiments the agent is an anti-angiogenic agent, e.g., a VEGF
receptor inhibitor such as an antibody or other binding moiety that
binds to a VEGF receptor and inhibits it by, e.g., blocking binding
of VEGF; an antibody or other binding moiety that binds to one or
more members of the VEGF family; an agent comprising a soluble VEGF
receptor extracellular domain (e.g., VEGF-Trap). In some
embodiments the agent enhances the expression of ICAM-1, ICAM-2, or
VCAM-1 on endothelial cells. In some embodiments the agent binds to
an endogenous molecule that may inhibit migration of immune system
cells, e.g., lymphocytes, across the endothelial barrier. Examples
of such molecules include endothelin B receptor. In some
embodiments the agent is an endothelin B receptor inhibitor. In
some embodiments the endothelin B receptor inhibitor is an antibody
or other binding moiety that binds to the endothelin B receptor. In
some embodiments the endothelin B receptor inhibitor is small
molecule such as BQ-788. In some embodiments the cells are immune
system cells, which in some embodiments may be sortagged with a
second agent, wherein the second agent has anti-tumor activity,
such as an immune checkpoint inhibitor. In some embodiments the
cells are red blood cells that are sortagged with an agent that has
anti-tumor activity, such as an immune checkpoint inhibitor. In
some embodiments the cells are CAR cells.
[0403] In some aspects the invention provides compositions
comprising any two or more cells or cell populations described
herein, wherein at least one of the cells or cell populations
comprises a non-genetically engineered polypeptide having an agent
conjugated thereto by sortase. In some aspects the invention
provides methods comprising using any two or more cells or cell
populations described herein in the same method, e.g.,
administering any two or more cells or cell populations described
herein to a subject, wherein at least one of the cells or cell
populations comprises a non-genetically engineered polypeptide
having an agent conjugated thereto by sortase. All different
combinations are envisioned. In some embodiments the cells or cell
populations are of the same cell type, e.g., two or more red blood
cells or red blood cell populations conjugated with different
agents, or two or more T cells or T cell populations conjugated
with different agent. In some embodiments the cells or cell
populations are of different cell types, e.g., red blood cells and
lymphocytes (e.g., T cells), In some embodiments the two or more
cells or cell populations are individually of use for the same
purpose, e.g., anti-tumor therapy. In some embodiments the two or
more cells or cell populations may have an additive or synergistic
effect. In some embodiments a combination may comprise red blood
cells comprising a non-genetically engineered polypeptide that is
sortagged with a first agent in combination with immune system
cells, e.g., lymphocytes, PBMCs, NK cells, sortagged with the same
agent or a different agent. Without limiting the invention in any
way, certain embodiments useful for cancer therapy may comprise (1)
red blood cells that are sortagged with an immune checkpoint
inhibitor or angiogenesis inhibitor in combination with immune
system cells, e.g., T cells or PBMCs, sortagged with an agent
comprising a binding moiety that binds to a tumor antigen; (2) red
blood cells that are sortagged with an immune checkpoint inhibitor
or angiogenesis inhibitor in combination with CAR cells that are
sortagged with an agent that enhances immune system cell
infiltration into tumors; (3) red blood cells that are sortagged
with an immune checkpoint inhibitor or angiogenesis inhibitor in
combination with CAR cells that comprise a CAR that binds to a
first tumor antigen and are sortagged with an agent that binds to a
second tumor antigen.
[0404] A subject may be treated with various preparative regimens
prior to administration of sortagged cells. For example,
lymphodepletion of the patient before adoptive cell transfer, which
eliminates T regulatory cells and other lymphocytes, is a component
of many ACT regimens for cancer (Dudley M E, et al. Adoptive cell
transfer therapy following non-myeloablative but lymphodepleting
chemotherapy for the treatment of patients with refractory
metastatic melanoma. J. Clin. Oncol. 2005; 23:2346-2357). Such
lymphocytes might otherwise compete with the transferred cells for
homeostatic cytokines such as interleukin-7 (IL-7) and IL-15.
Lymphodepletion before ACT may use total body irradiation or
cytotoxic drugs to deplete the lymphoid compartment of patients.
The transferred T cells may be administered with appropriate growth
factors to stimulate their survival and expansion in vivo and/or
such growth factors may be administered separately to the patient
prior to or following administration of the cells. In some
embodiments, molecules capable of stimulating endogenous antigen
presenting cells, such as Toll-like receptor agonists may be
administered. In some embodiments both T cells and APCs are
administered.
[0405] In some embodiments cells may be sortagged with a detectable
label so that they can be detected in vivo or in a sample
subsequently removed from a subject.
[0406] In some aspects, methods of modulating the immune system of
a mammalian subject are provided herein. In some embodiments a
method of modulating the immune system comprises administering a
living mammalian cell to a mammalian subject, wherein a moiety
comprising an immunomodulator, antigen, or epitope, is conjugated
to a living mammalian cell using sortase. In some embodiments a
method of modulating the immune system comprises conjugating a
moiety comprising an immunomodulator, antigen, or epitope to a
living mammalian cell using sortase and administering the living
mammalian cell to a mammalian subject.
[0407] In some embodiments, modulating the immune system comprises
modulating one or more biological activities of one or more types
of immune system cells. In some embodiments, modulating the immune
system comprises modulating an immune response to an antigen. In
some embodiments, modulating an immune response to an antigen
comprises modulating one or more biological activities of one or
more types of immune system cells exposed to the antigen. In some
embodiments an immune response comprises migration, proliferation,
or activation of one or more types of immune system cells. In some
embodiments an immune response comprises development of immature
immune system cells into mature, functional cells. In some
embodiments an immune response comprises proliferation and/or
activation of helper (CD4+) T cells specific for an antigen. In
some embodiments an immune response comprises proliferation and/or
activation of cytotoxic (CD8+) T lymphocytes (CTLs) specific for an
antigen. In some embodiments an immune response to an antigen
comprises production of cytokines by, e.g., immune system cells
specific for the antigen. In some embodiments an immune response
comprises proliferation and/or activation of antibody-producing
cells (plasma cells) and/or production of antibodies by such cells,
wherein the antibodies bind to an antigen. In some embodiments an
immune response comprises production of memory T and/or B cells
that are capable of providing a rapid immune response to an antigen
upon subsequent exposure to the antigen that elicited their
production. In some embodiments modulating an immune response
comprises modulating any one or more biological activities of
immune system cells. In some embodiments modulating an immune
response to an antigen comprises modulating any one or more
biological activities of immune system cells that are specific for
the antigen. In some embodiments modulating an immune response to
an antigen modulates an immune response to an entity comprising the
antigen. For example, modulating an immune response to a
pathogen-derived antigen modulates the immune response to a
pathogen comprising the antigen or a cell expressing the antigen or
displaying the antigen at its surface. The term "pathogen-derived
antigen" encompasses any antigen that is naturally produced by
and/or comprises a polypeptide or peptide that is naturally
genetically encoded by a pathogen, e.g., any of the various
pathogens mentioned herein. In some embodiments a pathogen-derived
antigen is a polypeptide, a polysaccharide, a carbohydrate, a
lipid, a nucleic acid, or combination thereof that is naturally
produced by a pathogen. In some embodiments a pathogen-derived
antigen is naturally encoded by a pathogen and is produced by an
infected cell as a result of the introduction into the cell of the
pathogen's genetic material that encodes the antigen. In some
embodiments a pathogen-derived antigen is at least partly exposed
at the surface of a cell membrane, cell wall, or capsule. In some
embodiments a pathogen-derived antigen is a secreted virulence
factor of a pathogen. In some embodiments a pathogen-derived
antigen is an antigen that plays a role in entry of the pathogen
into a host cell. For example, the antigen may bind to a cell
surface molecule of a cell to be infected. In some embodiments a
pathogen-derived antigen is a toxin. In some embodiments a pathogen
may be an agent that rarely if ever causes disease in healthy,
immunocompetent individuals, but that causes disease in at least
some individuals who are susceptible, e.g., individuals who are
immunocompromised.
[0408] In some embodiments, modulating an immune response comprises
stimulating (enhancing, augmenting, eliciting) an immune response.
In some embodiments "stimulating" an immune response encompasses
causing development of an immune response, enhancing the capacity
of a subject to mount an immune response, or increasing an immune
response in a subject who is currently mounting an immune response.
In some embodiments enhancing the capacity of a subject to mount an
immune response results in a faster or more robust immune response.
In some embodiments an immune response is directed towards foreign
entities (e.g., pathogens), infected cells, cancer cells, or other
undesirable (e.g., deleterious) cells or substances that comprise
the antigen.
[0409] In general, a targeting moiety may comprise any of a variety
of different moieties, which may be obtained using any suitable
method. In some embodiments a targeting moiety comprises an
antibody, an antibody chain, an antibody fragment, an scFv, a VHH
domain, a single-domain antibody, protein, or an aptamer, wherein
the antibody, antibody chain, antibody fragment, scFv, VHH domain,
single-domain antibody, protein, or aptamer binds to the
target.
[0410] In some embodiments, methods disclosed herein of modulating
an immune response enhance an adaptive immune response against a
pathogen, infected cell, tumor cell, or other undesired cell or
substance. In some embodiments, methods disclosed herein of
modulating an immune response enhance an innate immune response
against a pathogen, infected cell, tumor cell, or other undesired
cell or substance. In some embodiments, methods disclosed herein of
modulating an immune response enhance both an adaptive immune
response and an innate immune response. In some embodiments,
methods disclosed herein enhance a T cell-mediated immune response,
e.g., against a pathogen such as a virus (e.g., HIV), bacterium
(e.g., Mycobacterium), fungus (e.g., Aspergillus) or parasite
(e.g., Plasmodium), or against a tumor cell or other undesired
cell. In some embodiments, methods disclosed herein enhance
cell-mediated cytotoxicity towards a pathogen, infected cell, or
tumor cell. For example, in some embodiments methods disclosed
herein enhance activity of CD8+ cytotoxic T cells against a
pathogen, infected cell, or tumor cell.
[0411] In some embodiments a composition comprises sortagged
mammalian cells, wherein the cells are sortagged with any moiety of
interest. In some embodiments a composition comprises mammalian
cells, sortase, and a sortase substrate. In some embodiments a
composition comprises up to about 10.sup.14 cells, e.g., about 10,
10.sup.2, 10.sup.3, 10.sup.4, 10.sup.5, 5.times.10.sup.5, 10.sup.6,
5.times.10.sup.6, 10.sup.7, 5.times.10.sup.7, 10.sup.8,
5.times.10.sup.8, 10.sup.9, 5.times.10.sup.9, 10.sup.10,
5.times.10.sup.10, 10.sup.11, 5.times.10.sup.11, 10.sup.12,
5.times.10.sup.12, 10.sup.13, 5.times.10.sup.13, or 10.sup.14
cells. In some embodiments the number of cells may range between
any two of the afore-mentioned numbers. In some embodiments a
composition further comprises a growth factor, cytokine, adjuvant
or costimulator. In some embodiments the one or more growth factors
or cytokines promotes maturation, survival, proliferation, or
activation of at least some of the cells. In some embodiments a
cytokine is IL-2. In some embodiments a cytokine is IL-7, IL-12,
IL-15, or IL-21. In some embodiments a cytokine is TNF-alpha. In
some embodiments a composition in which immune system cells are
cultured or maintained comprises an antibody or ligand of a T cell
receptor or portion thereof, e.g., an antibody to CD3. In some
embodiments a composition in which immune system cells are cultured
or maintained comprises one or more adjuvants. In some embodiments
a composition comprising immune system cells comprises one or more
adjuvants that induces expression of a costimulator by at least
some of the immune system cells. In some embodiments the one or
more adjuvants comprises a TLR ligand, PAMP or PAMP mimic, CD40
ligand, or anti-CD40 antibody. In some embodiments a composition in
which immune system cells are cultured or maintained comprises one
or more costimulators. In some embodiments a costimulator is
expressed at the surface of APCs, e.g., DCs. In some embodiments a
costimulator is soluble. In some embodiments a costimulator is
attached to a surface, e.g., a particle.
[0412] In some embodiments an immune response comprises maturation,
proliferation and/or activation of lymphocytes, e.g., CD4+ helper T
cells, that are specific for the antigen, i.e., that express
receptors (TCR, BCR) that bind to the antigen, e.g., with high
affinity. In some embodiments, cell activation results in increased
expression of one or more cytokine genes.
[0413] Cells, e.g., sortagged mammalian cells, may be characterized
or assessed, e.g., to determine whether they have one or more
properties of interest, to determine the effect of sortagging,
and/or to determine the effect of the cells when administered to a
subject. In some embodiments, cells may be assessed for secretion
of one or more cytokines, presence of a cell surface marker profile
characteristic of an activated state, and/or possession of one or
more functional activities such as cytotoxic activity. In some
embodiments effector function of lymphocytes (e.g., T cells) may be
demonstrated by IFN.gamma. secretion after co-culture with target
cells (e.g., tumor cells, infected cells, or cells that have been
loaded with or caused to express a target antigen). In some
embodiments effector function of lymphocytes may be demonstrated by
increased expression of CD107a (LAMP-1), which may serve as a
functional marker for the identification of CD8+ T cell and natural
killer cell activity (e.g., degranulation). Cytokine secretion may
be assessed using, e.g., ELISA assay, cytokine antibody arrays,
etc. In some embodiments, presence or proliferation of T cells with
specificity for a particular antigen may be assessed using
peptide-MHC multimers (e.g., dimers, tetramers, pentamers, etc.)
which can be used to identify or isolate T cells specific for the
peptide, or in some embodiments using CD1-lipid multimers, which
can be used to identify or isolate natural killer T cells specific
for the lipid. Methods for generating peptide-MHC or CD1-lipid
multimers are well known in the art.
[0414] A wide variety of assays are available to assess cell
viability and/or proliferation. For example, a cell membrane
integrity assay (e.g., ability to exclude a compound that is
generally excluded from viable cells, such as trypan blue or
7-amino actinomycin D (7-AAD), cellular ATP-based assay, a
mitochondrial reductase activity assay, calcein staining, a DNA
content assay using a nucleic acid dye, a cellular metabolism assay
such as resazurin (sometimes known as AlamarBlue, etc.), MTT, XTT,
and CellTitre Glo, etc., a protein content assay such as SRB
(sulforhodamine B) assay; nuclear fragmentation assays; cytoplasmic
histone associated DNA fragmentation assay; PARP cleavage assay;
TUNEL staining; annexin staining, CyQUANT.RTM. cell proliferation
assays (Life Technologies). In some embodiments cytotoxicity may be
assessed using a flow cytometry based assay, wherein a fluorochrome
or other detectable label is used to stain non-viable or viable
cells in a cell population, and the cells are subjected to flow
cytometry and quantified.
[0415] In some embodiments cytotoxicity (e.g., cytotoxic effect of
sortagged mammalian cells) may be assessed, if desired, using any
suitable assay. In some embodiments a chromium-51 (.sup.51Cr)
release assay may be used. In a .sup.51Cr assay, target cells are
loaded with .sup.51Cr and maintained under conditions in which
cytolysis may occur. The label may then be released from the target
cells by cytolysis. The label can be isolated by centrifuging the
samples and collecting the supernatants. Supernatants from
centrifugation can either be counted directly in a gamma counter,
or mixed with scintillation fluid or dried on a substrate
comprising a solid scintillant such as a LumaPlate.TM. and counted
in a scintillation counter. Cytotoxicity assays utilizing similar
principles to the chromium-51 release assay (loading cells with a
compound or compound precursor and detecting compound subsequently
released from lysed cells) may be used, such as the DELFIA.RTM.
cytotoxicity assay (Perkin Elmer). The DELFIA is based on loading
cells with an acetoxymethyl ester of a fluorescence enhancing
ligand. After the ligand has penetrated the cell membrane the ester
bonds are hydrolyzed within the cell to form a hydrophilic ligand,
which no longer passes through the membrane. After cytolysis the
released ligand is introduced to a europium solution to form a
fluorescent chelate. The measured signal correlates directly with
the amount of lysed cells. In some embodiments a cytotoxicity assay
is used to measure cell-mediated cytotoxicity, e.g., cytotoxic
activity of T cells or NK cells. In some embodiments the ability of
cells to lyse target cells expressing particular peptides or other
antigens may be tested using cells that have been loaded with or
caused to express such peptides or antigens as target cells. In
some embodiments a cytotoxic cell is characterized in that it
produces perforin, granzyme(s) (e.g., granzyme A, granzyme B,
granzyme 3/K), and/or granulysin. In some embodiments production of
such enzymes may be detected by flow cytometry. In some
embodiments, cytotoxicity may be observed using, e.g., time lapse
microscopy.
[0416] In some embodiments pro-apoptotic activity (e.g.,
pro-apoptotic effect of sortagged mammalian cells) may be assessed,
if desired, using any suitable assay. In some embodiments a TUNEL
assay, DNA fragmentation assay, Annexin V assay, caspase assay,
mitochondrial membrane potential assay, etc.
[0417] The ability of administered sortagged mammalian cells to
produce a useful therapeutic effect may be assessed using standard
methods for assessing the effect of therapies in the particular
disease that the cells are intended to treat. For example,
anti-tumor effect may be assessed in a variety of non-human animal
tumor models, e.g., xenograft models, non-human animals with tumors
that arise spontaneously or as a result of genetic engineering,
etc. In some embodiments tumor(s) may be removed from the body
(e.g., at necropsy) and assessed (e.g., tumors may be counted,
weighed, and/or size (e.g., dimensions) measured). In some
embodiments the size and/or number of tumors may be determined
non-invasively. For example, in certain tumor models, tumor cells
that are fluorescently labeled (e.g., by expressing a fluorescent
protein such as GFP) can be monitored by various tumor-imaging
techniques or instruments, e.g., non-invasive fluorescence methods
such as two-photon microscopy. The size of a tumor implanted or
developing subcutaneously can be monitored and measured underneath
the skin. Any of a wide variety of methods and/or devices known in
the art may be used to assess tumors in vivo in animals or in human
subjects. Tumor number, size, growth rate, or metastasis may, for
example, be assessed using various imaging modalities, e.g., 1,2,
or 3-dimensional imaging (e.g., using X-ray, CT scan, ultrasound,
or magnetic resonance imaging, etc.) and/or functional imaging
(e.g., PET scan) may be used to detect or assess lesions (local or
metastatic), e.g., to measure anatomical tumor burden, detect new
lesions (e.g., metastases), etc. In human subjects, objective
criteria such as the original or revised Response Evaluation
Criteria In Solid Tumors (RECIST) (Therasse P, et al. J Natl Cancer
Inst (2000) 92:205-16; Eisenhauer, E., et al., Eur J Cancer. (2009)
45(2):228-47) or in the case of lymphomas or leukemias, response
criteria described in Cheson B D, et al. J Clin Oncol 2007;
10:579-86, may be used. As will be appreciated, in a clinical
response may not be evident until a number of weeks or months after
therapy. For example, in the case of a response to immune
checkpoint inhibitor therapy, a regression in size of lesions may
be slower (e.g., delayed by about 6 months after initiation of
therapy) as compared with the timing that is typically seen in the
case of responses to standard chemotherapeutic agents. Therapeutic
effect against a pathogen may, e.g., be assessed based on symptoms
of infection in pathogen-exposed animals or human subjects, and/or
by detecting a reduction in the presence of pathogens or
pathogen-infected cells in body fluids (e.g., blood), tissues, or
organs in infected subjects that receive the therapy as compared
with controls.
[0418] Animal models exist for a variety of different autoimmune
diseases. For example, the Collagen Induced Arthritis (CIA) model
is a commonly used model that shares immunological and pathological
similarities to human rheumatoid arthritis. Arthritis is initiated
by intradermal injections of Collagen Type II (CII) emulsified in
Complete Freund's Adjuvant (CFA), e.g., in rodents, which causes an
immune response generating antibodies to CII. There is both a T
cell and B cell component to the pathology. Experimental autoimmune
encephalomyelitis is an inflammatory demyelinating disease of the
central nervous system (CNS) and is used as an animal model of
human CNS demyelinating diseases, including multiple sclerosis and
acute disseminated encephalomyelitis. EAE serves as a prototype for
T-cell-mediated autoimmune disease in general. EAE can be induced
in a number of species, including mice, rats, guinea pigs, rabbits
and primates. Commonly used antigens in rodents are spinal cord
homogenate (SCH), purified myelin, myelin protein such as MBP, PLP
and MOG, or peptides of these proteins. It may also be induced by
the passive transfer of T cells specifically reactive to these
myelin antigens. Therapeutic efficacy may be assessed based on,
e.g., clinical symptoms and signs, histopathology (e.g., lesions,
tissue destruction), presence of self-reactive T cells, etc. Animal
models of type I diabetes include, for example, non-obese diabetic
(NOD), BDC2.5 transgenic, and humanized mice such as
NOD..beta.2mnull.HHD mice, which lack murine-derived MHC I and
instead transgenically express human HLA-A2.1 molecules.
[0419] Cells may be administered in an effective amount, by which
is meant an amount sufficient to achieve a biological response or
effect of interest, e.g., reducing one or more symptoms or
manifestations of a disease or condition or modulating an immune
response. In some embodiments a composition administered to a
subject comprises up to about 10.sup.14 cells, e.g., about
10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6, 10.sup.7, 10.sup.8,
10.sup.9, 10.sup.10, 10.sup.11, 10.sup.12, 10.sup.13 or 10.sup.14
cells, or any intervening number or range. In some embodiments
between about 10.sup.5 and about 10.sup.12 cells are administered.
In some embodiments between about 10.sup.5-10.sup.8 cells and about
10.sup.11-10.sup.13 cells are administered. In some embodiments a
subject receives a single dose of cells. In some embodiments a
subject receives multiple doses of cells, e.g., between 2 and 5,
10, 20, or more doses, over a course of treatment. In some
embodiments a dose or total cell number may be expressed as
cells/m.sup.2 or cells/kg. For example, a dose may be about
10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6, 10.sup.7, 10.sup.8,
10.sup.9, or 10.sup.10 cells/m.sup.2 or cells/kg any intervening
number of range. In some embodiments a course of treatment lasts
for about 1-2 months, 2-6 months, 6-12 months, or more, e.g.,
indefinitely or until the subject is no longer in need of
treatment. In some embodiments a subject may be treated about every
2-6 weeks. One of ordinary skill in the art will appreciate that
the number of cells, doses, and/or dosing interval may be selected
based on various factors such as the weight, surface area, and/or
blood volume of the subject, the condition being treated, response
of the subject, etc. The exact number of cells required may vary
from subject to subject, depending on factors such as the species,
age, weight, sex, and general condition of the subject, the
severity of the disease or disorder, the particular cell(s), the
identity and activity of agent(s) conjugated to the cells, mode of
administration, concurrent therapies, and the like. It will be
understood that the amount may be decided by the attending
physician within the scope of sound medical judgment. In some
embodiments both sortagged and non-sortagged cells may be
administered.
[0420] In some embodiments one or more compounds is also
administered once or more to the subject in addition to
administering cells. In some embodiments a compound is administered
at least once prior to and/or at least once after administration of
the cells. In some embodiments a cytokine is administered, wherein
the cytokine is capable of enhancing survival, proliferation,
maturation, activation, or activity of immune system cells. In some
embodiments the cytokine is IL-2. In some embodiments the cytokine
is IL-7, IL-12, IL-15, or IL-21. In some embodiments an adjuvant is
administered. In some embodiments the adjuvant is capable of
inducing APCs to express a costimulator. In some embodiments the
adjuvant and/or cytokine is administered in the same composition as
the cells. In some embodiments the adjuvant, cytokine, and/or cells
are administered in different compositions.
[0421] In general, cells may be administered using any suitable
route of administration. In some embodiments cells are administered
to the circulatory system, e.g., by infusion. In some embodiments
cells are administered intravenously. In some embodiments cells are
administered to or in the vicinity of a tumor or a site that may
harbor tumor cells (e.g., a site from which a tumor was removed or
rendered undetectable by treatment or to which a tumor is prone to
metastasize), site of infection, or site of potential infection
(e.g., a break in the skin such as a wound, indwelling device,
surgical site, etc.), or any site at which an effect, e.g., a
therapeutic effect, is desired. In some embodiments cells are
administered into or in the vicinity of an organ that is affected
by a condition for which the cells have a therapeutic effect. In
some embodiments the organ is one in which a tumor is present or
from which a tumor has been removed or to which a tumor is prone to
metastasize. In some embodiments the tumor is a primary tumor. In
some embodiments the tumor is a metastatic tumor. One of ordinary
skill in the art will be aware that certain tumor types are prone
to metastasize to particular organs, i.e., they metastasize to
those organs commonly or at least more frequently than to many or
most other organs. For example, breast tumors are prone to
metastasize to bone, liver, lung and brain; colorectal cancers are
prone to metastasize to the liver and lungs. In some embodiments
the organ is the first, second, third, or fourth most common
organto which the particular tumor type that the individual has
metastasizes. In some embodiments the tumor is a metastasis. In
some embodiments cells are administered into the portal vein or
hepatic artery to treat a liver condition, e.g., a liver cancer
(e.g., hepatocellular carcinoma) or liver infection. In some
embodiments cells are administered into the pancreatic artery to
treat a condition affecting the pancreas, e.g., pancreatic cancer.
In some embodiments cells are administered into the peritoneal
cavity to treat a condition affecting the peritoneum, such as a
tumor. Primary peritoneal cancer is a cancer of the cells lining
the peritoneum and is a form of mesothelioma. The peritoneal cavity
is a common site of ovarian cancer spread or recurrence. Peritoneal
metastases may arise from a variety of primary cancer, such as
gastrointestinal cancers. In some embodiments cells are
administered into the pleural space or thoracic cavity to treat a
condition affecting the lungs or pleura, such as a lung cancer or
pleural mesothelioma. In some embodiments cells are administered
into the spinal canal to treat a condition affecting the brain or
meninges such as a tumor. In some embodiments cells are
administered intraocularly to treat a condition affecting the eye,
such as a tumor. In some embodiments cells are administered into or
in the vicinity of an organ in which an infection has been detected
or suspected of being present Presence of an infection in an organ
may be suspected e.g., based at least in part on symptoms or signs
experienced or exhibited by the subject, detection of the
infectious agent or a component thereof (such as DNA, RNA, protein)
in a sample obtained from the organ, or known propensity of the
infectious agent to infect organs of that type. In some
embodiments, "in the vicinity" of a site or organ refers to within
a location outside the organ or site and within 1 cm, 2 cm, 3 cm, 4
cm, 5 cm from the edge of the organ or site. In some embodiments,
"in the vicinity" of a site or organ refers to administration into
a blood vessel that supplies the organ or site, either within the
organ or site or at a location no more 5 cm, 10 cm, 15 cm, 20 cm,
or 25 cm away from the point where the blood vessel (or one or more
blood vessels that arise from the blood vessel) enter the organ or
site. In certain embodiments cells may be introduced into a vessel
that transports blood out of or away from the organ. In some
embodiments, such administration may be useful to target tumor
cells that arise from a tumor within the organ and enter the
circulatory or lymphatic system.
[0422] In some embodiments, focused ultrasound (FUS) in the
presence of a microbubble contrast agent may be used to deliver
sortagged cells to the brain, e.g., for treatment of a tumor or
infection in the brain using immune system cells targeted to an
antigen expressed in the tumor, by infected cells, or by an
infectious agent. The FUS disrupts the blood brain barrier (BBB),
facilitating delivery of cells to the brain. In some embodiments
the immune system cells are sortagged CAR cells. In some
embodiments the cells are sortagged with a targeting moiety.
[0423] Cells may be administered in any physiologically acceptable
vehicle. A vehicle compatible with cell viability and not causing
adverse effects when administered to a subject may be selected by
the ordinary skilled artisan. In some embodiments a vehicle
comprises water, appropriate salt concentration, and may comprise a
physiologically compatible buffer substance such as HEPES.
[0424] In some embodiments immune system cells are administered to
a subject in need of prophylaxis or in need of treatment of an
existing cancer or in need of delaying, inhibiting, or preventing
recurrence of cancer. In some embodiments at least some of the
introduced cells (or their descendants) mount an immune response
against the cancer or against cancer cells remaining in or arising
in the body, wherein the cancer or cancer cells comprise the tumor
antigen. In some embodiments at least some of the introduced cells
(or their descendants) stimulate maturation, proliferation, and/or
activation of at least some endogenous immune system cells of the
subject, e.g., endogenous T cells, wherein the endogenous immune
system cells mount an immune response against the cancer or against
cancer cells remaining in or arising in the body, wherein the
cancer or cancer cells comprise the tumor antigen.
[0425] In some embodiments a method comprises identifying an
antigen expressed by a tumor for which a subject is in need of
treatment. The tumor or cells obtained from the tumor can be
analyzed for expression of tumor antigens using standard methods
such as immunohistochemistry, flow cytometry, etc. In some
embodiments, immune system cells are sortagged with an agent
comprising a targeting moiety that binds to the antigen. The immune
system cells and/or descendants thereof are subsequently
administered to the subject. In some embodiments immune system
cells are obtained from a subject prior to treatment of the subject
with chemotherapy or radiation. At least some of the immune system
cells may be stored for future use in producing one or more cell
preparations to be administered to the subject.
[0426] In some embodiments a subject, e.g., a subject to whom
sortase-modified cells are administered, is immunocompetent, e.g.,
the subject has a normally functioning immune system. In some
embodiments a subject is immunocompromised. A subject may be
immunocompromised for any of a variety of reasons. Such reasons may
include, e.g., age (e.g., infants or elderly individuals), genetic
immunodeficiency disorders affecting one or more components of the
innate and/or adaptive immune system, diseases such as cancer or
infections that affect the immune system such as HIV infection,
treatment with an immunsuppressive or cytotoxic drug, e.g., for
cancer (e.g., cancer chemotherapy) or to prevent or inhibit
transplant rejection or to treat an autoimmune disease.
Immunosuppresive agents include, e.g., cytotoxic or cytostatic
drugs, such as a variety of chemotherapeutic drugs used in the
treatment of cancer, various drugs administered to reduce the
likelihood of transplant rejection or to treat autoimmune diseases.
Examples include, e.g., glucocorticoids, immunophilin-interacting
agents such as rapamycin or rapamycin analogs, TNF alpha
antagonists, etc.). In some embodiments a subject is at increased
risk of infection as compared with a normal, average healthy
individual, due, e.g., to hospitalization, surgery, chronic disease
(e.g., diabetes, cancer, chronic obstructive pulmonary disease,
cystic fibrosis), indwelling medical device (e.g., catheter, IV
line), implant or prosthesis (e.g., heart valve replacement),
physical trauma, burn, malnourishment, etc. In some embodiments
sortase-modified cells are used to induce or augment an immune
response in a subject who has undergone, is undergoing, or will
undergo chemotherapy or radiation therapy. In some embodiments a
subject is at increased risk of infection because the subject is
less than about 1 year of age or is over about 60, 65, 70, 75, or
80 years of age.
[0427] In some embodiments, modulating an immune response comprises
inhibiting the immune response. As used herein, "inhibiting" an
immune response encompasses preventing or delaying development of
an immune response to an antigen in a subject not currently
exhibiting such response or reducing the intensity of a current or
potential future immune response. In some embodiments an immune
response is an unwanted immune response, e.g., an immune response
that is deleterious to the subject in whom it occurs. In some
embodiments, an unwanted immune response is directed against self
tissues or cells, transplanted tissue or cells, non-living
materials introduced into the body for diagnostic or therapeutic
purposes, or an allergen.
[0428] In some embodiments an unwanted immune response is an immune
response that is excessive or inappropriately prolonged, such that
it is deleterious to the subject. For example, an immune response
directed against an antigen derived from a pathogen that has
infected a subject may initially be beneficial in terms of
controlling the pathogen but may be too intense or prolonged, such
that it causes tissue damage to the subject (e.g., cell-mediated or
antibody-mediated tissue damage) or symptoms due to excessive
cytokine release.
[0429] An unwanted immune response may be mounted by a subject
against a transplanted tissue or organs or cells, such as blood
cells, stem cells, blood vessel, bone marrow, solid organ (e.g.,
heart, lung, kidney, liver, pancreas), skin, intestine, or cells
derived from any of the foregoing. For example, pancreatic tissue,
e.g., pancreatic islets, or isolated pancreatic beta cells, may be
transplanted from a donor into a subject in need of treatment of
diabetes, e.g., type I diabetes. In some embodiments a transplant
(also termed a "graft") comprises allogeneic cells or tissues
(i.e., the donor and recipient are different individuals from the
same species). In some embodiments a transplant comprises
xenogeneic cells or tissues (i.e., the donor and recipient are of
different species). The immune response may be directed, e.g.,
against one or more donor antigens, e.g., histocompatibility
proteins (e.g., major or minor histocompatibility proteins) of the
donor. An immune response directed against a graft may be referred
to as "rejection". Rejection may result in damage to the graft,
which may reduce its function, may lead to graft failure, and may
ultimately require removal of the graft. In some embodiments
sortagged mammalian cells comprise regulatory T cells conjugated
with a moiety that targets them to cells, tissues, or organs at
risk of or exhibiting evidence of rejection, e.g., acute graft
rejection.
[0430] An unwanted immune response may comprise graft-versus-host
disease (GvHD). GvHD may occur, for example, after an allogeneic
stem cell transplant. In GvHD, grafted donor immune cells or their
descendants recognize the recipient (e.g., recipient's cells) as
foreign and mount an immune response thereto, e.g., a T
cell-mediated immune response. Allogeneic hematopoietic stem cell
transplantation can be a curative treatment in a variety of
hematopoietic disorders, immunodeficiencies, and leukemias and
finds use in treatment of a variety of cancers when chemotherapy
has ablated the patient's immune system. GvHD can be a
life-threatening complication of such transplants. In some
embodiments sortagged mammalian cells may be used to treat, e.g.,
prophylactically, GvHD. In some embodiments sortagged mammalian
cells comprise regulatory T cells conjugated with a moiety that
targets them to cells, tissues, or organs at risk of or exhibiting
evidence of GvHD. In some embodiments, regulatory T cells may be
modified using sortase, e.g., with a targeting moiety that targets
them to graft-derived immune cells and/or to sites at which such
immune cells are active. Mesenchymal stem cells have been reported
to have a variety of beneficial effects in treatment of
graft-versus host disease (GvHD). For example, they have been
reported to reduce proliferation of graft derived T-cells, inhibit
alloreactive T-cell responses and support hematopoietic stem cell
(HSC) engraftment. In some embodiments, MSCs may be modified using
sortase, e.g., with a targeting moiety that targets them to
graft-derived T cells and/or to sites at which such T cells are
active. In some embodiments MSCs may be conjugated using sortase
with a moiety that targets them to cells, tissues, or organs at
risk of or exhibiting evidence of GvHD. In some embodiments the
regulatory T cells and/or MSCs are targeted to the skin, liver, or
gastrointestinal tract. In some embodiments the regulatory T cells
and/or MSCs may be conjugated with an agent, e.g., a cytokine, that
exerts an inhibitory effect on donor immune system cells involved
in GvHD. In some embodiments the regulatory T cells and/or MSCs may
be derived from the same donor as the original grafted cells.
[0431] In some embodiments an unwanted immune response comprises an
immune response to an autoantigen (also referred to as a self
antigen), e.g., in a subject suffering from an autoimmune disease.
Such inappropriate immune responses may involve self-reactive T
cells, autoantibodies, or both. One of ordinary skill in the art
will be aware of various autoantigens involved in particular
autoimmune diseases. Autoimmune diseases include, for example,
acute disseminated encephalomyelitis, alopecia areata,
antiphospholipid syndrome, autoimmune hepatitis, autoimmune
myocarditis, autoimmune pancreatitis, autoimmune polyendocrine
syndromes, autoimmune uveitis, inflammatory bowel disease (Crohn's
disease, ulcerative colitis), type I diabetes mellitus (e.g.,
juvenile onset diabetes), multiple sclerosis, scleroderma,
ankylosing spondylitis, sarcoid, pemphigus vulgaris, pemphigoid,
psoriasis, myasthenia gravis, systemic lupus erythemotasus,
rheumatoid arthritis, juvenile arthritis, psoriatic arthritis,
Behcet's syndrome, Reiter's disease, Berger's disease,
dermatomyositis, polymyositis, antineutrophil cytoplasmic
antibody-associated vasculitides (e.g., granulomatosis with
polyangiitis (also known as Wegener's granulomatosis), microscopic
polyangiitis, and Churg-Strauss syndrome), scleroderma, Sjogren's
syndrome, anti-glomerular basement membrane disease (including
Goodpasture's syndrome), dilated cardiomyopathy, primary biliary
cirrhosis, thyroiditis (e.g., Hashimoto's thyroiditis, Graves'
disease), transverse myelitis, and Guillane-Barre syndrome.
Examples of certain autoantigens that are involved in some of these
diseases are discussed below. In some embodiments sortagged
mammalian cells comprise regulatory T cells conjugated with a
moiety that targets them to cells, tissues, or organs at risk of or
exhibiting evidence of damage in an autoimmune disease.
[0432] In some embodiments, a method for inducing tolerance
comprises generating tolerogenic DCs, e.g., DCs that either delete
autoreactive T cells or induce regulatory T (Treg) cells, e.g.,
CD4+CD25-Foxp3+ regulatory T cells. In some embodiments, a method
results in reduction in the number and/or activity of Th17 cells.
In some embodiments tolerogenic DCs are generated in vitro and
administered to a subject. In some embodiments tolerogenic DCs are
generated by a method comprises exposing DCs, e.g., immature DCs,
in vitro, to an agent comprising (a) a targeting moiety that binds
to a DC cell surface protein and (b) an antigen, wherein the
antigen comprises a self-antigen or allergenic antigen. In some
embodiments inhibiting the immune response e.g., induction of
tolerance or a tolerogenic state, is achieved by using a suitable
concentration or amount of the agent and/or exposing cells or
subjects to appropriate cytokines. In some embodiments targeting an
antigen to DCs in the absence of an effective amount of an adjuvant
inhibits the immune response to the antigen that would otherwise
occur and thereby results in increased tolerance to the antigen. In
some embodiments a method of inhibiting an immune response
comprises administering to a subject an agent comprising a
targeting moiety that binds to DCs and an antigen, wherein the
antigen comprises a self-antigen or allergenic antigen. In some
embodiments the antigen is one to which the subject has previously
exhibited or continues to exhibit or is at risk of exhibiting an
unwanted, e.g., deleterious, immune response. In some embodiments
the agent is administered without administering an effective amount
of an adjuvant. For example, the agent may be administered in a
composition that is substantially free of adjuvants.
[0433] In some embodiments, a method for inducing or promoting
tolerance comprises generating modified mammalian Treg cells
("Tregs") using sortase and administering the cells to a subject.
As known in the art, Tregs are capable of regulating, e.g.,
inhibiting, immune responses by, for example, suppressing effector
T cells. In some embodiments Tregs are modified by conjugating a
targeting moiety to their surface using sortase, wherein the
targeting moiety targets the Tregs to an organ, tissue, or site in
the body of a subject where regulation, e.g., inhibition, of an
immune response is desired. In some embodiments, the organ, tissue,
or site is a graft. In some embodiments the Tregs are generated by
obtaining Tregs from a subject and modifying the Tregs ex vivo
using sortase. In some embodiments Tregs obtained from a subject
are expanded ex vivo prior to modification by sortase. In some
embodiments modified Tregs may be contacted with a graft ex vivo
prior to transplanting the graft into a receipient. The Tregs may
infiltrate the graft and inhibit development or limit the extent of
an immune response after transplant. In some embodiments Tregs may
be administered together with the graft and/or after the
transplant. Tregs may be administered locally at or near the site
of transplant and/or by introducing the cells into the bloodstream
or lymphatic system. In some embodiments at least one cytokine is
administered to the subject one or more times in combination with
Tregs. A cytokine and Tregs may be administered at the same site,
different sites, or both. A cytokine may be administered prior to,
at the same time as, and/or after administration of the Tregs. If
administered at the same time, the Tregs and cytokine(s) may be in
the same composition as the Tregs or in a different composition. In
some embodiments a cytokine is a cytokine that has
anti-inflammatory properties, e.g., IL-2, IL-10, transforming
growth factor-.beta. (TGF-.beta.), IL-27, IL-35 or IL-37. Such
anti-inflammatory properties may comprise, e.g., inhibiting
formation, maturation, expansion or activity of effector immune
system cells; stimulating formation, maturation, expansion or
activity of regulatory T cells, etc.
[0434] Tregs may be isolated and, if desired, expanded ex vivo
using methods known in the art (see, e.g., and references therein
for examples of such methods). Tregs may be identified based on a
cell surface marker expression pattern of CD4+CD25+CD127lo. In some
embodiments Tregs are characterized by high-level expression of
CD25, FoxP3, CTLA-4, GITR, and CD62L and a very low or undetectable
expression of CD127.
[0435] In some embodiments inhibiting an unwanted immune response
comprises stimulating an immune response against one or more
cellular components of the unwanted immune response. For example,
in some embodiments an immune response directed against
self-reactive immune system cells, e.g., self-reactive T cells, is
stimulated. In some embodiments an immune response directed against
immune system cells at least in part responsible for an
immune-mediated disorder, e.g., allergy, is stimulated. In some
embodiments an immune response directed against one or more
cellular components of the unwanted immune response at least in
part eliminates such cells, resulting in a reduction or inhibition
of the unwanted immune response.
[0436] In some embodiments a composition, e.g., a composition to be
used to induce tolerance in a subject, is substantially free or
essentially free of any one or more substances, e.g., any one or
more particular adjuvant(s), e.g., any one or more of the adjuvants
or classes of adjuvants mentioned above or known in the art. In
some embodiments the concentration or amount of adjuvant present,
if any, is ineffective to enhance an immune response. In some
embodiments the concentration or amount of adjuvant is less than or
equal to 1%, 5%, 10%, 15%, 20%, or 25% of the concentration or
amount that would be effective to stimulate an immune response,
e.g., an amount that would be used by one of ordinary skill in the
art seeking to generate or enhance an immune response against an
antigen, e.g., in a vaccine. In some embodiments a composition is
substantially free or essentially free of any one or more
particular adjuvant(s), e.g., any one or more of the adjuvants or
classes of adjuvants mentioned above or known in the art.
[0437] In some embodiments a method comprises identifying an
antigen to which a subject is allergic or reactive (e.g.,
self-reactive) and conjugating an agent comprising the antigen to a
mammalian cell using sortase. In some embodiments the cell is
capable of inducing tolerance to the antigen. In some embodiments
the cell is a Treg cell. In some embodiments the cell is
administered to a subject in need of treatment of an allergy or in
need of inhibition of immune response against the antigen. In some
embodiments identifying an antigen comprises administering a test
dose of one or more antigens to the subject, e.g., performing a
skin test. In some embodiments identifying comprises determining
the response of the subject to a test dose of one or more allergens
or antigens. In some embodiments, if the response to an allergen is
abnormally intense, the antigen is identified as one to which the
subject is allergic or self-reactive. In some embodiments the
subject harbors self-reactive T cells or B cells comprising a TCR
or BRC that recognizes the antigen. In some embodiments the subject
produces antibodies that bind to the antigen. In some embodiments a
method comprises determining whether a subject produces antibodies
that bind to an allergenic antigen or self-antigen. In some
embodiments a sample comprising cells or serum from a subject is
tested against a panel of candidate allergenic antigens or
autoantigens in order, e.g., to identify one or more allergenic
antigens or self-antigens at least in part responsible for causing
an allergy or autoimmune disease.
[0438] In some embodiments cells are sortagged with an antigen to
which the subject is allergic or self-reactive. In some embodiments
at least some of the cells are administered to the subject. In some
embodiments the cells promote or induce tolerance to the antigen.
For example, in some embodiments, a method for inducing or
promoting tolerance comprises generating modified mammalian cells
using sortase, wherein the cells are not genetically engineered for
sortagging, and administering the cells to a subject in need
thereof, e.g., a subject suffering from or at risk of an autoimmune
disease. The mammalian cells may be sortagged with any autoantigen
or allergen of interest or fragment thereof. In some embodiments
the mammalian cells may be obtained from the subject or from an
immunocompatible donor. In some embodiments the mammalian cells
comprise lymphocytes, PBMCs, splenocytes, or red blood cells. In
some embodiments the antigen comprises a T cell epitope. In some
embodiments the T cell epitope is a CD4+ epitope. In some
embodiments the T cell epitope is a CD8+ epitope. Cells may be
sortagged with an agent comprising a single epitope or multiple
epitopes, which may be from the same or different antigens. In some
embodiments multiple populations of cells, sortagged with agents
comprising different epitopes, may be administered. In some
embodiments a subject may be tested to identify autoantibodies and
determine which antigen(s) they react with. An agent (or agents)
comprising one or more epitopes from those particular antigens may
be conjugated to cells using sortase, for subsequent administration
to the subject. In some embodiments, inducing tolerance to an
autoantigen may reduce the clinical severity (e.g., reduce the
severity of one or more symptoms), reduce the rate of progression,
reduce the number of flare-ups, induce a remission, reduce the
level of one or more biomarkers or other indicators of the disease,
or otherwise show evidence of beneficial effect.
[0439] In some embodiments an autoimmune disease to be treated is
multiple sclerosis (MS). MS is an inflammatory demyelinating
disease of the CNS characterized by the formation of multiple
discrete inflammatory lesions and focal demyelination in
perivascular and periventricular sites of CNS white matter
(Nylander, A. & Hafler, D. A. (2012) J. Clin. Invest. 122,
1180-1188). These demyelinating lesions are marked by infiltration
of activated mononuclear cells and are associated with the
appearance of neurologic deficits. MS is believed to be an
autoimmune disorder caused at least in part by T cells specific for
immunodominant self-epitopes of myelin and other CNS antigens. It
is thought that such autoreactive T cells migrate into the CNS and
undergo re-activation upon T cell antigen recognition of endogenous
CNS epitopes and secrete pro-inflammatory cytokines and chemokines
which recruit inflammatory macrophages and other leukocytes from
the blood to initiate focal demyelination and CNS dysfunction. In
some embodiments the autoantigen is a myelin protein of the central
nervous system (CNS) such as myelin basic protein (MBP),
proteolipid protein (PLP), or myelin oligodendrocyte glycoprotein
(MOG). In some embodiments mammalian cells, e.g., RBCs or PBMCs,
are sortagged with an agent comprising any of these proteins or an
agent, e.g., a peptide, comprising an epitope found in any of these
proteins, and administered to a subject in need of treatment for
MS. In some embodiments the peptide comprises MBP amino acids
89-99.
[0440] In some embodiments an autoimmune disease to be treated is
type I diabetes (T1D). In some embodiments cells, e.g., RBCs or
PBMCs, are sortagged with an agent that comprises insulin (INS),
islet-specific G6Pase catalytic subunit-related protein (IGRP),
heat shock protein 60 (HSP60), islet cell antigen 512 (IA-2), or
other islet cell antigens, or an agent, e.g., a peptide, that
comprises an epitope of any of these proteins. For example, the
antigen may comprise insulin B amino acids 9-23. In some
embodiments the cells may be administered to a subject in need of
treatment for T1D. In some embodiments the cells may be
administered to a subject with T1D who is to receive or has
received a transplant comprising islet cells, e.g., isolated islet
cells, islets, or pancreatic tissue comprising islets.
[0441] In some embodiments an autoimmune disease to be treated is
an autoimmune blistering skin disease, which term refers to a group
of diseases characterized by autoantibodies against structural
components of the skin. Further details regarding these diseases
and particular autoantigens and epitopes implicated in their
pathogenesis may be found in Otten, J V, et al., Curr Mol Med.
January 2014; 14(1): 69-95, and references cited therein. In
pemphigus vulgaris (PV) autoantibodies react mainly with desmoglein
3 (Dsg3) alone and/or in combination with desmoglein 1 (Dsg1). In
bullous pemphigoid (BP) autoantibodies frequently target two
hemidesmosomal proteins, BP180 (collagen XVII) and BP230, but may
also target other proteins such as plectin and a6 integrin. In
mucous membrane pemphigoid autoantibodies target several
autoantigens of the dermal-epidermal junction, including BP180,
BP230, laminin 332, .alpha.6.beta.4 integrin, and collagen VII.
Autoimmunity to collagen VII is typically associated with the skin
blistering disease epidermolysis bullosa acquisita (EBA), but also
occurs occasionally in patients with systemic lupus erythematosus
or inflammatory bowel disease. Anti-p200 pemphigoid is an
autoimmune subepidermal blistering disease, characterized by
autoantibodies against a 200-kDa protein (p200) of the epidermal
basement membrane, which has been identified as the laminin yl
chain. In some embodiments cells, e.g., RBCs or PBMCs, are
sortagged with an agent comprising Dsg1, Dsg3, BP180, BP230,
laminin .gamma.1, collagen VII, plectin, a6 integrin, laminin 332,
or .alpha.6.beta.4 integrin or an agent, e.g., a peptide, that
comprises an epitope found in Dsg1, Dsg3, BP180, BP230, laminin
.gamma.1, collagen VII, plectin, .alpha.6 integrin, laminin 332, or
.alpha.6.beta.4 integrin. In some embodiments the epitope is in the
ectodomain of the relevant protein. The cells may be administered
to a subject in need of treatment for the relevant autoimmune
blistering skin disease, SLE, IBD, or other autoimmune disease.
[0442] In some embodiments an autoimmune disease to be treated is
an inflammatory bowel disease, e.g., Crohn's disease and ulcerative
colitis. Autoantibodies against exocrine pancreas (PAb) have been
reported to be pathognomonic markers of Crohn's disease (CD). For
example, the glycoproteins CUZD1 and GP2 are targets of PAb in
patient with Crohn's disease. In some embodiments cells, e.g., RBCs
or PBMCs, are sortagged with an agent comprising CUZD1 or GP2 or an
agent, e.g., a peptide, that comprises an epitope found in CUZD1 or
GP2. The cells may be administered to a subject in need of
treatment for Crohn's disease.
[0443] In some embodiments an autoimmune disease to be treated is
an inflammatory arthritis, e.g., rheumatoid arthritis (RA).
Autoantibodies to type II collagen (CII), heat shock proteins such
as the immunoglobulin binding protein (BiP), citrullinated peptides
(anti-CCP) and other citrullinated proteins such as vimentin and
fillagrin are found in patients with RA In some embodiments cells,
e.g., RBCs or PBMCs, are sortagged with an agent comprising type II
collagen (CII), heat shock proteins immunoglobulin binding protein
(BiP), human chondrocyte glycoprotein 39, citrullinated peptides
(anti-CCP) and citrullinated proteins such as citrullinated
fillagrin or citrullinated vimentin or an agent, e.g., a peptide,
that comprises an epitope found in any of these. The cells may be
administered to a subject in need of treatment for an inflammatory
arthritis, e.g., rheumatoid arthritis.
[0444] In some embodiments mammalian cells are sortagged with an
agent that is capable of inhibiting or reducing the effect of
(neutralizing) a toxic substance that may be present in the body of
a subject, e.g., in the blood. A toxic substance is a substance
that causes or is capable of causing death, injury, damage, or
other physiological disturbance to organisms when a sufficient
quantity is introduced into or onto or absorbed by a living
organism. Toxic substances include those substances recognized as
such in the art. The action of a toxic substance may be by chemical
reaction or other activity on the molecular scale. In some
embodiments a toxic substance exerts its effects when present in
the blood or when transported via the circulatory system to one or
more locations in the body. The agent may be, e.g., an antibody, at
least a portion of a receptor that binds to the toxic substance, or
any other binding agent that binds to the toxic substance.
[0445] As used herein, a "toxin" is a toxic substance produced
within living cells or organisms. In some embodiments a toxin may
be produced by or genetically encoded by a microbe, e.g., a
pathogen and/or may be produced in the body as a result of
infection by a pathogen. Toxins may be produced, e.g., by bacteria,
fungi, plants, protozoa, and parasites. In some embodiments a toxin
is an exotoxin, i.e., it is released, e.g., secreted, by cell(s)
that produce it. Examples of toxins include AB. toxins, e.g.,
AB.sub.1 toxins, AB.sub.5 toxins. As used herein, an "AB.sub.1
toxin" is a toxin that comprises an A subunit and a B subunit. It
will be understood that a subunit, e.g., an A subunit, may be
cleaved to produce two polypeptide chains, which may be linked by
one or more disulfide bonds. Diphtheria toxin (C. diphtheriae) is
an exemplary AB1 toxin. Heparin-binding epidermal growth
factor-like growth factor serves as a receptor for diphtheria
toxin. Pseudomonas exotoxin A, another bacterial AB.sub.1 toxin,
utilizes the low density lipoprotein receptor-related protein, also
known as the a2-macroglobulin receptor to enter cells. AB.sub.1
toxins also include certain type II ribosome inactivating plant
toxins such as ricin, abrin, cinnanomin, viscumin, ebulin, and
nigrin b (Hartley, M R & Lord, J M, Cytotoxic
ribosome-inactivating lectins from plants, Biochim Biophys Acta,
1701 (1-2): 1-14, 2004; Xu H, et al., Cinnamomin.about.a versatile
type II ribosome-inactivating protein. Acta Biochim Biophys Sin
(Shanghai) 36(3): 169-76). A complete AB.sub.5 toxin complex
contains six protein units, five B subunits that are similar or
identical in structure and a single A subunit. The A subunit (or a
portion thereof) of an AB.sub.5 toxin is the portion of the complex
responsible for toxicity. The B subunits form a pentameric
(five-membered) ring, into which the A subunit extends and is held.
The B subunits may protect the A subunit and mediate binding to
cells. Examples of AB.sub.5 toxins (and names of bacteria that
produce them) include, e.g., Campylobacter jejuni enterotoxin (C.
jejuni), cholera toxin (V. cholerae), heat-labile enterotoxins LT
and LT-II (Escherichia coli), pertussis toxin (B. pertussis), shiga
toxin (S. dysenteriae), shiga-like toxin (also known verotoxin)
SLT1 and SLT2 (certain E. coli). Other toxins of interest include,
e.g., Botulinum neurotoxin (C. botulinum), tetanus neurotoxin (C.
tetani), and the large clostridial toxins known as Toxin A and
Toxin B (C. difficile).
[0446] In some embodiments a toxic substance, e.g., a toxin, is a
cytolysin, which term refers to substances, e.g., proteins and
lipids, that cause lysis of cells, e.g., by damaging their cell
membrane. In some embodiments a toxin is a hemolysin, which term
refers to substances that cause lysis of red blood cells Hemolysins
can be identified by their ability to lyse red blood cells in
vitro. In some embodiments a hemolysin may, in addition to
affecting red blood cells, also affect other cells, such as
leukocytes. In some embodiments a toxin is a pore-forming toxin
(PFT). PFTs, which include certain cytolysins, can be divided into
the following subcategories: alpha-pore-forming toxins (e.g.,
cytolysin A of E. coli, aerolysin (Aeromonas hydrophila), and
Clostridial Epsilon-toxin), beta-pore-forming toxins (e.g.,
.alpha.-hemolysin, Panton-Valentine leukocidin, Vibrio cholerae
cytolysin, and S. aureus gamma-hemolysin, Clostridium perfringens
enterotoxin). Other cytolysins include, e.g., anthrax toxin,
cholesterol-dependent cytolysins such as pneumolysin, and small
pore-forming toxins such as gramicidin A.
[0447] In certain embodiments a toxin is a protein, e.g., an
enzyme, that degrades or directly damages host cell molecules or
tissues. Examples, include, e.g., hyaluronidase, proteases,
coagulases, lipases, deoxyribonucleases. In certain embodiments a
toxic substance is a superantigen or superantigen-like protein.
Superantigens (SAgs) are a class of antigens that cause
non-specific activation of T-cells resulting in polyclonal T cell
activation and massive cytokine release. T cell activation is
believed to result from SAg-mediated cross-linking of major
histocompatibility complex (MHC) class II antigens and T-cell
receptors (TCRs). SAgs contain distinct domains capable of binding
to MHCII and TCR. SAgs can be produced by pathogenic microbes
(e.g., certain bacteria) or may be endogenous and produced in
response to infection by pathogenic microbes (e.g., certain
viruses). Many SAgs are exotoxins, a number of which are produced
by the Gram-positive organisms Staphylococcus aureus and
Streptococcus pyogenes. SAgs are the causative agents in toxic
shock syndrome, among other disorders. Many superantigens share a
common architecture that is also shared by the superantigen-like
proteins (SSL), another group of bacterial virulence factors.
Certain Sags and SLLs are discussed in Fraser J D, Proft T. The
bacterial superantigen and superantigen-like proteins. Immunol Rev.
2008; 225:226-43.
[0448] In some embodiments a toxin is an endotoxin, which refers to
toxins that are typically not released from the cells that produce
them (except in the case of destruction or damage to the cell,
e.g., destruction of the bacterial cell wall). Examples of
endotoxins are lipopolysaccharide (LPS) or lipooligosaccharide
(LOS), found in the outer membrane of various Gram-negative
bacteria. The toxic effects of endotoxins on vertebrate organisms
are mediated by their interaction with receptors on immune system
cells, which results in synthesis and/or release of immune
mediators such as cytokines, nitric oxide, and eicosanoids,
excessive amounts of which can damage the organism.
[0449] Further information regarding certain toxins discussed above
and many others may be found, e.g., in Alouf, J E & Popoff, M
R, (eds.) The Comprehensive Sourcebook of Bacterial Protein Toxins,
Third Edition, Academic Press, 2006; Schmitt, M J & Schaffrath,
R (eds.) Microbial Protein Toxins, Topics in Current Genetics 1 1,
Berlin, N.Y.: Springer-Verlag, 2005; Pro ft, T. (ed.) Microbial
toxins: molecular and cellular biology, Norfolk, England: BIOS
Scientific, c2005.
[0450] In some embodiments a toxic substance may not be directly
toxic but may enhance the effect of a toxic substance or may be
required for its activity. For example, the substance may mediate
entry of a toxic substance into cells. In some embodiments a
substance may be converted into a toxic substance in the body.
[0451] In some embodiments a subject may be accidentally or
deliberately exposed to a toxic substance. In some embodiments a
toxic substance may be a pesticide (e.g., an insecticide or
herbicide), a chemical used or produced in industrial processes,
etc. In some embodiments a toxic substance may be a therapeutic
agent that has been administered to a subject in an excessive
amount and/or has been administered to a subject who has reduced
(e.g., below normal) capacity to metabolize or excrete the
substance.
[0452] In some embodiments a toxic substance may be produced by a
subject's own cells, e.g., in response to infection or as part of a
disease process. For example, certain cytokines and inflammatory
mediators produced in response to infection or injury or in certain
diseases may have damaging effects when present in excessive
amounts. Examples include, e.g., pro-inflammatory cytokines such as
interferon gamma, TNF-alpha, IL-1 (e.g., IL-1.beta.), IL-6, and
IL-17, and inflammatory mediators such as leukotrienes.
[0453] In some embodiments a toxic substance is a virulence factor
or component thereof. Virulence factors are substances or
structures produced or encoded by pathogens (bacteria, viruses,
fungi, protozoa, or multicellular parasites) that play a role in
establishing and/or maintaining an infection. A virulence factor
may permit or increase the ability of a pathogen to achieve one or
more of the following: colonization of a niche in the host,
immunoevasion, evasion of the host's immune response,
immunosuppression, entry into and/or exit out of cells or cellular
compartments (if the pathogen is an intracellular one during at
least part of its life cycle), obtain nutrition from the host.
Virulence factors include, e.g., pathogen-produced toxins, adhesive
molecules (e.g., adhesins), molecules that stimulate endocytosis,
immunoglobulin-binding proteins, proteases that degrade
immunoglobulins or other host cell molecules that play a role in
the immune response, bacterial capsule, which may inhibit
phagocytosis of the bacteria by host immune cells, complement
inactivating molecules, structures such as pili or fimbriae. In
some embodiments a virulence factor is a biofilm component. In some
embodiments a virulence factor is encoded by a plasmid or
bacteriophage.
[0454] Cells may be sortagged with any suitable moiety capable of
binding to and/or inhibiting a toxic substance or virulence factor.
In some embodiments an inhibitor of a toxic substance or virulence
factor may bind to the substance or structure and thereby prevent
it from acting on or interacting with its target, may inactivate
the substance or structure (e.g., by cleaving it), etc. It will be
understood that inhibition may be partial or complete. Examples of
suitable inhibiting moieties include, e.g., proteins, aptamers, or
other moieties that are capable of binding to the substance or
structure, enzymes that are capable of cleaving the substance or
structure, etc. In some embodiments an inhibitor of a toxic
substance comprises a naturally occurring receptor for the
substance or a fragment or variant of the receptor, wherein the
variant or fragment is sufficient for binding the substance. A
variety of agents capable of inhibiting toxic substances are known
in the art and may be incorporated into agents that are used to
sortag mammalian cells.
[0455] In some embodiments, cells may be sortagged with an agent
capable of binding to a pathogen or pathogen-secreted molecule,
e.g., any pathogen or pathogen-secreted molecule of interest. In
some embodiments the pathogen is one that is typically present in
the blood of a vertebrate, e.g., mammalian, host during all or part
of the pathogen's life cycle. In some embodiments the
pathogen-secreted molecule is secreted into the blood or gains
entry into the blood of a vertebrate, e.g., mammalian, host
infected by the pathogen.
[0456] In some embodiments living mammalian cells may be sortagged
with a detectable label. The sortagged cells may be administered to
a subject and subsequently detected in vivo or in a sample obtained
from the subject. The detectable label may be selected to permit in
vivo detection, e.g., by an imaging technique such as ultrasound,
PET scan, MRI, fluorescence detection (e.g., near infrared). In
some embodiments living mammalian cells may be sortagged with
detectable label and a targeting moiety capable of targeting the
cells to a target of interest, e.g., in the body of a subject. The
cells become attached to the target, thereby concentrating the
detectable moiety at the target. Detection of the detectable label
may allow detection of the target. The target may be any molecule,
cell, or structure in the body of a subject. In some embodiments
the target may be a pathogen, pathogen component, or
pathogen-secreted substance. In some embodiments the target may be
a toxic substance.
[0457] In some embodiments living mammalian cells sortagged with a
detectable label may be administered to a subject together with
other cells of the same type that are not sortagged. The
distribution and/or concentration of sortagged cells may be
representative of the distribution and/or concentration of the
administered population. Such sortagged cells may serve as tracking
agents, e.g., detection and/or quantification of the sortagged
cells provides an indication of the distribution and/or
concentration of the administered population. For example, when
administering cells for adoptive immunotherapy it may be of
interest to determine where such cells localize and/or their
average residence time in the body. In some embodiments a
population of cells may be isolated based on any one or more
criteria or properties of interest, such as a particular cell
surface marker expression pattern, gene expression profile,
functional activity, or based on their having been generated
through or subjected to a particular protocol or exposed to
particular agents. Cells may be sortagged with a detectable label
and mixed with one or more other cell populations (which may or may
not be sortagged, e.g., with different detectable label(s) or other
moietie) or administered to a subject. The label(s) may be used to
detect the cell in vitro or in vivo. For example, it may be of
interest to monitor cell migration, cell-cell physical
interactions, cell division, or cell distribution.
[0458] In some embodiments, sortase-modified mammalian may be used
in regenerative medicine. Regenerative medicine as used herein
refers to therapies that comprise replacing or regenerating
mammalian, e.g., human, cells, tissues or organs to improve
function, e.g., to restore or establish normal function and/or
structure, by administering cells to the subject and/or by
administering biologically active substances that act on endogenous
cells or tissues to promote their healing or regeneration. Examples
of regenerative medicine therapies include using implanted
cartilage cells (e.g., chondrocytes) to restore cartilage,
strategies to remuscularize the injured heart, e.g., using adult
stem cells, pluripotent stem cells, or cardiomyoctes, ex vivo
production of tissues or organs which may then be implanted into
subjects, among others.
[0459] Sortase-modified mammalian cells may be used in regeneration
of any of a wide variety of tissues and organs. Tissues and organs
of interest include, e.g., cartilage, bone, heart, heart valve,
blood vessel, esophagus, stomach, liver, gallbladder, pancreas,
intestines, rectum, anus, endocrine gland (e.g., thyroid,
parathyroid, adrenal, endocrine portion of pancreas, e.g., islets
of Langerhans), skin, hair follicle, tooth, gum, lip, nose, mouth,
thymus, spleen, skeletal muscle, smooth muscle, joint, brain,
spinal cord, peripheral nerve, ovary, fallopian tube, uterus,
vagina, mammary gland, testes, vas deferens, seminal vesicle,
prostate, penis, pharynx, larynx, trachea, bronchi, lungs, kidney,
ureter, bladder, urethra, eye (e.g., conjunctiva, retina, retinal
pigment epithelium, cornea), or ear (e.g., organ of Corti). In some
embodiments, a tissue is an epithelial layer, e.g., an epithelial
layer lining the interior of a hollow organ. Regenerative medicine
encompasses tissue engineering, i.e., the use of living cells
seeded on a natural or synthetic extracellular substrate to create
implantable structures, e.g., parts of an organism.
[0460] In some embodiments sortase is used to conjugate a moiety to
a cell that is subsequently administered to a subject in need of
regeneration, e.g., of a tissue or organ. In some embodiments the
cell may be administered into, adjacent to, or near (e.g., within 5
cm, 10 cm, 20 cm, or 25 cm) a tissue or organ whose regeneration is
desired. For example, if cardiac regeneration is desired, the cells
may be administered to the heart. In some embodiments the cell may
be administered into the circulatory system. In some embodiments a
moiety comprises a targeting moiety binds to a target at a location
in the body at which regeneration is desired. In some embodiments
the target may be a cell-type specific marker expressed by cells in
the tissue or organ to be regenerated. The targeting moiety may
enhance attachment of the administered cell to a site where
regeneration is desired or may enhance integration of the
administered cell into a tissue or organ whose regeneration is
desired. In some embodiments a moiety may promote survival,
proliferation, or functional activity of the administered cell. In
some embodiments the moiety may promote survival, proliferation, or
functional activity of cells found in a tissue or organ whose
regeneration is desired. In some embodiments the moiety may promote
migration of circulating or resident adult stem cells to a tissue
or organ whose regeneration is desired or may promote retention,
functional integration, and/or differentiation of such adult stem
cells. Examples of moieties that may be useful for regenerative
medicine include, e.g., growth factors, survival factors, cell
adhesion molecules,
[0461] In some embodiments sortase-modified cells (e.g., conjugated
with an agent that promotes cell survival, proliferation,
functional activity, or tissue integration) may be used in the
production of tissues or organs ex vivo, which may then be
implanted into a subject. For example, they may be used, optionally
in combination with unmodified cells, to seed two-dimensional or
three-dimensional scaffolds (sometimes termed "matrices" or
"constructs") ex vivo, or they may be used to provide appropriate
stimulatory signals (e.g., growth or survival signals) to cells
that are used in the production of tissues or organs ex vivo. A
scaffold promote cell-biomaterial interactions, cell adhesion, and
ECM deposition, may permit sufficient transport of gases,
nutrients, and regulatory factors to allow cell survival,
proliferation, and differentiation, and may in some embodiments
biodegrade at a rate that approximates the rate of tissue
regeneration under the culture conditions or in vivo conditions of
interest. In some embodiments a scaffold is comprised of materials
that provoke no or minimal degree of inflammation or toxicity in
vivo. Scaffolds may comprise, e.g., decellularized structures such
as decellularized blood vessels or organs, or may comprise
synthetic scaffolds such as those produced using various synthetic
polymers. Polymer scaffolds may be porous and/or biodegradable in
certain embodiments. Examples of polymers or polymer compositions
of natural origin of use in forming scaffolds include collagen,
gelatin, fibrin, hyaluronic acid, alginate, and chitosan. Synthetic
polymers of use in forming scaffolds include polyglycolide,
poly(L-lactic acid), poly(l-lactide-co-glycolide),
poly(.epsilon.-caprolactone). It will be appreciated that
derivatives, copolymers, and blends of natural and/or synthetic
polymers may be used. Decellularization can provide an acellular,
three-dimensional biologic scaffold that can be seeded with
selected cell populations. A scaffold may be composed of ECM
proteins typically found in the body. The three-dimension
architecture of a scaffold may be similar to that of the original
tissue or organ, which may thus confer appropriate mechanical and
physical properties. Agents conjugated to sortase-modified cells
may, e.g, promote cell attachment to a scaffold, may promote
cell-cell adhesion, may promote cell survival, proliferation,
and/or differentiation.
VII. Kits and Services for Sortagging Mammalian Cells and/or Other
Eukaryotic Cells
[0462] In some aspects, the invention provides kits useful for
generating sortagged mammalian cells, wherein the cells are not
genetically engineered for sortagging. In some embodiment a kit
comprises (i) a sortase polypeptide, a nucleic acid or vector that
encodes a sortase polypeptide, or a cell line that expresses
sortase polypeptide; and (ii) one or more items useful in the
preparation, characterization, and/or purification of sortagged
mammalian cells. In s one or more items may be any of the items
that are described herein with regard to methods of preparing,
characterizing, and/or purifying sortagged living mammalian cells
that are not genetically engineered for sortagging. In some
embodiments the one or more additional items are selected from: (a)
an agent comprising a transamidase recognition sequence; (b)
mammalian cells that are not genetically engineered for sortagging;
(c) a liquid composition, or components thereof, suitable for use
as a reaction buffer in which to sortag living mammalian cells; (d)
one or more reagents useful for separating sortagged cells from
sortase; and (e) a control substance. In some embodiments a kit
comprises instructions for preparing sortagged mammalian cells that
are not genetically engineered for sortagging. In some embodiments
instructions may be made available separately from the kit. For
example, instructions may be provided or accessed via the Internet,
e.g., on the World Wide Web ("web"). In some embodiments the agent
comprising a transamidase recognition sequence comprises a binding
moiety that binds to a tumor antigen. The agent may be used, e.g.,
to sortag cells that are to be administered to a subject in need of
treatment for a tumor that expresses the TA. In some embodiments,
any of the afore-mentioned kits may not comprise a sortase. For
example, the agent comprising a transamidase recognition sequence
may be provided alone or together with one or more of the
afore-mentioned additional items.
[0463] In some embodiments, a kit comprises a plurality of distinct
agents each comprising a transamidase recognition sequence, wherein
distinct agents each comprise a different binding moiety. In some
embodiments, each of a plurality of different binding moieties
present in agents in the kit binds to a different tumor antigen. A
health care facility, e.g., a hospital, that treats patients in
need of treatment for tumors may obtain such a kit and may use it
on site to sortag cells to be administered to the patient. The
particular agents to be conjugated to cells may be selected from
those present in the kit, based at least in part on results of
analyzing expression of tumor antigens on a particular patient's
tumor. In some embodiments, a kit comprises at least 5, 10, 15, 20,
25, 30, 40, or 50 different agents, e.g., up to about 100, 200,
500, or more agents, each comprising a binding moiety that binds to
a different TA. Any one or more of the TAs mentioned above, in any
combination, may be represented by agents in the kit. In some
embodiments sortase is provided in the kit, optionally together
with any one or more of the additional agents mentioned above. In
some embodiments sortase may be provided separately.
[0464] In some aspects, the invention provides methods in which
sortagging of eukaryotic cells, e.g., mammalian cells, may be
performed as a service. An organization or individual that performs
sortagging for other organizations and/or individuals, e.g., upon
request, may be referred to as a "sortagging service provider". In
some embodiments a sortagging service provider may be in the
business of providing services, e.g., research services,
manufacturing services, to the pharmaceutical industry,
biotechnology industry, biomedical research community, etc. In some
embodiments a sortagging service provider may have a website and
may offer sortagging services on its website. In some embodiments a
sortagging service provider receives a request (also referred to as
an "order") for sortagged mammalian cells from a requestor, which
may be any organization or individual that seeks to obtain
sortagged mammalian cells. An organization may be a for-profit
organization, a non-profit organization, a company, a corporation,
a contract research organization, a research institution, an
academic institution, etc. A request may be submitted, transmitted,
received, and/or stored at least in part electronically or in
electronic format. In some embodiments a request is submitted via a
webpage and transmitted via the Internet. A request may indicate
the type of cells and/or the agent(s) to be conjugated to the
cells. In some embodiments a request may indicate one or more
additional specification such as the number of cells, manufacturing
conditions (e.g., research grade, clinical grade (e.g.,
GMP-compliant), storage conditions, shipping conditions, etc. In
some embodiments the requestor may provide the cells and/or may
provide one or more agent(s) to be conjugated to the cells or one
or more moieties to be incorporated into agent(s) to be conjugated
to cells. For example, in some embodiments a requestor may provide
cells that originate from and/or are designated for administration
to a particular subject. The sortagging service provider performs
or arranges for sortagging according to the request and supplies
sortagged cells, e.g., to the requestor or as indicated by the
requestor. A sortagging service provider may perform the sortagging
and/or providing itself and/or through arrangements with other
organizations or individuals. In some embodiments a sortagging may
be performed for a fee, under a contract, or according to a quote.
A sortagging service provider may offer one or more ancillary
services that may facilitate or support or may be useful in
connection with generating or using sortagged mammalian cells.
Examples include, e.g., cell expansion services, preparation of
agents to be conjugated to mammalian cells, characterization of
sortagged cells (e.g., assays of cell function or biological
activity of an agent conjugated to the cells). In some embodiments,
a sortagging service provider analyzes a tumor sample from a
subject who is in need of treatment for a tumor and identifies one
or more tumor antigens expressed by cells of the tumor, to which
therapeutic cells are to be targeted. The sortagging service
provider may sortag cells to be administered to the subject with
one or more targeting moieties that bind to the tumor antigen(s).
The sortagged cells may be transported to a location where they are
administered to the subject.
[0465] In some embodiments, any of the above-mentioned kits and
services may be used for the sortagging of non-mammalian eukaryotic
cells, e.g., cells of non-mammalian vertebrate or invertebrate
animals, fungal cells, or protozoal cells, in addition to or
instead of mammalian cells. Thus, where the discussion of kits and
services herein refers to mammalian cells, the invention provides
embodiments that encompass non-mammalian eukaryotic cells.
VIII. Certain Aspects and Embodiments
[0466] Sortase-modified cells, e.g., sortase-modified mammalian
cells, can be used to treat a wide variety of different diseases
and disorders. In some embodiments sortase-modified cells
conjugated with a therapeutic agent may be used to treat any
disease or condition for which the unconjugated therapeutic agent
is of use. Such methods of treatment are an aspect of the
invention.
[0467] In some embodiments sortase-modified cells may be
administered prophylactically, e.g., to a subject who does not
exhibit signs or symptoms of the disease or disorder for which the
cells are indicated (but may be at increased risk of developing the
disorder or is expected to develop the disease or disorder). In
some embodiments sortase-modified cells are administered to a
subject who has developed one or more signs or symptoms of the
disease or disorder, e.g., the subject has been diagnosed as having
the disease or disorder. Optionally, a method comprises diagnosing
a subject as having a disease or disorder for which
sortase-modified cells are an appropriate treatment.
[0468] One of ordinary skill in the art will be aware of various
indications for therapeutic agents that may be conjugated to
mammalian cells. For example, interferons have a variety of uses,
e.g., in the treatment of autoimmune diseases (e.g., multiple
sclerosis) and certain infectious diseases (e.g., certain viral
infections) and (often in combination with chemotherapy and
radiation) as a treatment for many cancers.
[0469] In some embodiments sortagged mammalian cells or other
sortagged eukaryotic cells (e.g., sortagged eukaryotic
microorganisms) administered for treatment of a disease may be used
in combination with any other therapy useful in treating the
disease. When entities are administered "in combination" they may
be administered in the same composition (if compatible) or in
different compositions in various embodiments. When administered in
different compositions any order of treatment is contemplated.
Administration of or more doses of sortagged cells may be
interspersed with administration of one or more doses of one or
more other agents. Successive doses may be administered at times
separated by time intervals of minutes, hours, days, weeks, or
months. In some embodiments at least one dose of sortagged cells is
administered within no more than 1, 2, 3, 4, 6, 8, 10, 12, 16, 20,
22, 24, 26, 30, 36, 42, 48, or 52 weeks before or after
administration of one or more doses of a different therapeutic
entity, e.g., any therapeutic agent useful in treating the disease.
In some embodiments, a time interval between a dose of cells and a
dose of a cytotoxic or anti-proliferative drug is selected so as to
avoid significant effect, e.g., cytotoxic or anti-proliferative
effect, of the cytotoxic or anti-proliferative drug on the
cells.
[0470] In some embodiments sortagged mammalian cells administered
for treatment of cancer may be used in combination with a
chemotherapy drug and/or radiation therapy. The cells may be
administered separately from the drug. In some embodiments cycles
of drug and/or radiation therapy may be interspersed with cycles of
cell therapy. In certain embodiments it is contemplated to
administer sortagged cells in addition to any standard cancer
treatment regimen, e.g., any standard chemotherapy and/or radiation
regimen or instead of one or more components of such a regimen. In
some embodiments a subject in need of treatment of cancer may
undergo surgery to remove at least a portion of the tumor. The
surgery may remove the entire tumor (to the extent the tumor is
detectable) or may reduce the size of the tumor but not remove the
entire tumor (e.g., if the tumor is too extensive to make complete
surgical removal advisable or if attempting such surgical removal
is otherwise not advisable within the judgement of the skilled
artisan). In some embodiments sortagged cells may be administered
to the subject one or more times prior to surgery, one or more
times during surgery, and/or one or more times after surgery. The
sortagged cells may reduce the size of a tumor and/or may eliminate
tumor cells that were not removed during surgery (which may be
located at the site of surgery or may have disseminated to other
location(s) in the body.
[0471] Erythropoiesis stimulating agents such as EPO are of use to
treat anemia, which may result from a variety of causes. For
example, the anemia may be an anemia of chronic disease, anemia
associated with medications (e.g., cancer chemotherapy), radiation,
renal disease (e.g., diabetes), infectious diseases, or blood loss.
Colony stimulating factors such as G-CSF, GM-CSF, and/or M-CSF may
be used to treat leukopenia, e.g., neutropenia and/or lymphopenia,
which may result, e.g., from medications (e.g., cancer
chemotherapy), radiation, infectious disease, or blood loss.
[0472] Neurotrophic factor proteins may be used, e.g., to treat
neurodegenerative diseases (e.g., amyotrophic lateral sclerosis,
Huntington disease, Alzheimer disease, Parkinson disease), central
or peripheral nervous system injury.
[0473] Interleukins are of use to modulate the immune response for
a wide variety of purposes, e.g., to stimulate an immune response
against an infectious agent or cancer or to limit the intensity
and/or duration of innate and/or adaptive immune responses.
Interleukins may be of use in treatment of autoimmune diseases,
sepsis, or other conditions in which an aberrant or overactivated
immune response can be deleterious.
[0474] Diseases caused by viruses, gram-positive or gram-negative
bacteria, mycobacteria, fungi, or parasites are of interest in
certain embodiments. For example, immune system cells may be
sortagged with an agent that binds to such viruses, bacteria,
fungi, or parasites, or may be sortagged with an agent that binds
to mammalian cell infected by such viruses, bacteria, fungi, or
parasites. Exemplary viruses, bacteria, fungi, and parasites are
discussed above.
[0475] In general, a sortagged eukaryotic cell may be used for any
purpose in which it is useful to have an agent attached to the
surface of such cell. In some embodiments, the agent comprises a
detectable label, thus facilitating detection of the sortagged
eukaryotic cell. In some embodiments the cell is a microorganism
that spends at least part of its life cycle as an intracellular
parasite of a mammalian or avian host. Examples of such
microorganisms include a variety of fungi and protozoa. In some
embodiments, an intracellular parasite that has been labeled with
sortase is contacted with a host cell, e.g., a mammalian or avian
cell, in vitro. Processes such as attachment of the microorganism
to the cell, entry of the microorganism, intracellular movement of
the microorganism, or other activities of the microorganism may be
monitored by detecting the label. In some embodiments a sortagged
microorganism is of use in a method of identifying a candidate
therapeutic agent for treating a disease caused by the
microorganism. For example, in some embodiments, a candidate
compound is contacted with the cell, and the ability of the
candidate compound to inhibit one or more such activities is
assessed. In some embodiments, if the candidate compound inhibits
one or more such activities, the candidate compound is identified
as a candidate therapeutic agent for treating a disease caused by
the microorganism.
[0476] In some embodiments, a sortagged microorganism, e.g., a
sortagged eukaryotic microorganism, is used to deliver an agent to
a target cell, e.g., a tumor cell, tumor-associated cell, or
pathogen-infected cell. In some embodiments the microorganism is
sortagged with the agent, with a targeting moiety that binds to a
molecule on the target cell, or both. In some embodiments the
microorganism is one that is naturally capable of invading the
target cell. For example, the microorganism may be an intracellular
parasite (during at least one of its life cycle stages), e.g., an
apicomplexan parasite such as T. gondii, a Trypanosomatid, a
Plasmodium, a fungus such as Histoplasma capsulatum or Cryptococcus
neoformans, and the target cell may be a vertebrate cell, e.g., a
mammalian cell, e.g., a human cell, that is susceptible to invasion
by the microorganism. Those of ordinary skill in the art will be
aware of various strains of microorganisms and suitable methods of
obtaining and propagating microorganisms. In some embodiments the
microorganism is sortagged with a targeting moiety that binds to a
tumor antigen and increases the binding of the microorganism to a
cell that expresses the TA at its surface and, in some embodiments,
increases subsequent invasion of the microorganism. In embodiments
in which the microorganism is sortagged with a targeting moiety,
the targeting moiety may be of any of the various types of binding
moieties described herein. In some embodiments the targeting moiety
comprises a single chain antibody (e.g., an scFv) or single domain
antibody. For example, in some embodiments the targeting moiety
comprises a VHH, some embodiments the targeting moiety comprises a
single chain antibody (e.g., an scFv) or single domain antibody.
For example, in some embodiments the binding moiety comprises a
VHH. In some embodiments the microorganism is sortagged with an
agent comprising a substance that is toxic to the target cell upon
delivery to the surface of the cell or to the interior of the
target cell. In some embodiments the microorganism is sortagged
with an agent comprising a substance that is toxic to the target
cell upon contact with the target cell surface. In some embodiments
the toxic substance is a chemotherapy drug, e.g., a small molecule
chemotherapy drug, a toxin, a protein comprising a cytolytic domain
such as granzyme or perforin, a pro-apoptotic agent such as a
protein comprising a pro-apoptotic domain. In some embodiments the
microorganism is genetically engineered to produce one or more
molecules, e.g., a substance that is toxic to the target cell, a
cytokine, a costimulator, a targeting moiety. In some embodiments
the microorganism secretes the molecule or expresses it at its cell
surface. In some embodiments the microorganism is genetically
engineered to lack expression or activity of one or more endogenous
gene products. One or ordinary skill in the art will be aware of
appropriate methods and vectors useful for creating genetically
engineered microorganisms. In some embodiments, a strain, e.g., a
T. gondii strain, that has a deficient non-homologous end joining
pathway may be used (Fox B A, Eukaryot Cell. 2009; 8(4):520-9). For
example, the strain may lack expression of a functional KU80
protein or homolog thereof, e.g., due to a disruption or deletion
in the gene that encodes KU80 or homolog thereof. In some
embodiments the microorganism is avirulent and/or can be
effectively eliminated by treating the subject with an appropriate
therapeutic agent.
[0477] In some embodiments the target cell is not a tumor cell or
pathogen-infected cell. The target cell may be, e.g., a normal,
healthy cell or an abnormal cell. In some embodiments the
microorganism may be sortagged with an agent that modulates one or
more biological activities or properties of the normal or abnormal
cell, e.g., in a way that is beneficial to a subject to whom the
cell is administered or in whom the cell exists. In some
embodiments, the cell may be an immune system cell, and the agent
may be, e.g., an immunomodulator. In some embodiments the
microorganism is genetically engineered to produce a substance,
e.g., a protein. The substance may be a therapeutic agent, enzyme,
or any other substance the production of which is desired. In some
embodiments the substance modulates one or more biological
activities or properties of the cell. In some embodiments the cell
is affected by a disease, and the substance ameliorates the effect
of the disease on the cell. In some embodiments the microorganism
is sortagged with a targeting moiety that binds to the normal or
abnormal cell.
[0478] In some embodiments the microorganism is an attenuated
strain. "Attenuated" refers to a strain that has reduced virulence
relative to a wild type strain or parental strain from which an
attenuated strain is generated. An attenuated strain may be
weakened and/or less robust compared to a wild type strain or
parental strain (i.e., a strain from which the attenuated strain
was derived). In some embodiments an attenuated strain is
avirulent. An attenuated strain may arise naturally and be
identified by testing the virulence of the strain in a test system
(e.g., in test cells or a test animal) or may be generated by man
by, e.g., passaging the organism in a host or host cells that are
not a natural host or host cell of the microorganism, by
mutagenesis and selection, by irradiation, by exposure to a
chemical agent, or by engineering. In some embodiments an
attenuated strain has substantially reduced or absent ability to
inflict damage on a host or host cell, has substantially reduced or
absent ability to replicate or complete one or more stages of its
life cycle in a particular host or host cell (e.g., humans or human
cells), and/or has substantially reduced or absent transmissibility
from one host to another as compared to a wild type strain or as
compared to a parental strain. In some embodiments an attenuated
strain has at least a 10-fold, 10.sup.2-fold, 10.sup.3-fold,
10.sup.4-fold, 10.sup.5-fold, 10.sup.6-fold, 10.sup.7-fold, or
10.sup.8-fold reduced ability to replicate as compared to a wild
type strain or as compared to a parental strain. In general,
replication ability may be measured using any suitable method. In
some embodiments, replication ability may be measured as number of
new individual organisms produced or amount of DNA synthesized by
the organism and its descendants. In some embodiments an attenuated
strain has at least a 10-fold, 10.sup.2-fold, 10.sup.3-fold,
10.sup.4-fold, 10.sup.5-fold, 10.sup.6-fold, 10.sup.7-fold, or
10.sup.8-fold reduced ability to cause death of a host or host cell
as compared to a wild type strain or as compared to a parental
strain. In some embodiments an attenuated strain is metabolically
active. In some embodiments an attenuated strain retains ability to
invade host cells, e.g., mammalian host cells, e.g., human host
cells. If desired, invasion may be quantified using an invasion
assay. One of ordinary skill in the art will be aware of suitable
assays. The strain's ability to invade host cells may or may not be
equivalent to that of a wild type strain or parental strain. In
some embodiments the invasion ability is at least 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90%, or 100% of that of a wild type strain
or parental strain. In some embodiments an attenuated strain may
have a normal or near normal level of virulence under certain
conditions or in certain hosts but is avirulent or has
substantially reduced virulence under other conditions, such as
those existing in vivo in a subject, e.g., a human subject. In some
embodiments, for example, an attenuated strain may be auxotrophic
for one or more nutrients. The strain may replicate normally under
conditions in which the nutrient is available (e.g., supplied in a
culture medium) but may be unable to replicate under in vivo
conditions in which the amount of the nutrient is insufficient to
support replication. In some embodiments the nutrient is a
precursor for nucleotide biosynthesis, e.g., a precursor for
synthesis of a purine or pyrimidine. Attenuated strains are known
in the art. For example, attenuated T. gondii strains are described
in U.S. Pat. Pub. Nos. 20100203085 and/or 20120045477, which also
describe methods and vectors useful for genetic engineering of T.
gondii. In some embodiments an attenuated T. gondii strain has a
defect in de novo pyrimidine synthesis, pyrimdine salvage, and/or
transport of pyrimidine bases or nucleosides. For example, one or
more genes in the de novo pyrimidine synthesis pathway, pyrimidine
salvage pathway, or a transporter of pyrimidine bases or
nucleosides may be disabled, e.g., by targeted insertional
mutagenesis. In some embodiments the gene encodes carbamoyl
phosphate synthetase II, aspartate transcarbamylase,
dihydroorotase, dihydroorotase dehydrogenase, orotate
phosphoribosyltransferase, or orotidine 5'-monophosphate
decarboxylase, uridine phosphorylase (UP), uracil
phosphoribosyltransferase, or purine nucleoside phosphorylase. In
some embodiments an attenuated strain is the cps strain of T.
gondii, which has a knockout of the gene that encodes carbamoyl
phosphate synthetase II and exhibits uracil auxotrophy and
extremely reduced virulence (B. A. Fox, D. J. Bzik. Nature. 2002;
415:926-929). The cps strain invades and replicates normally in
vitro if uracil is supplied in the culture medium. As uracil is not
present at an adequate concentration to support replication of the
cps strain in mammals or mammalian cells, it invades mammalian host
cells normally but does not replicate and exhibits greatly
decreased virulence. In some embodiments a subject to whom a
sortagged attenuated microorganism is administered is an individual
who is infected with a non-attenuated strain of the microorganism.
A variety of microorganisms have been used or proposed for use as
live, attenuated vaccines, e.g., for protection against infection
by non-attenuated strains of the microorganism, are known in the
art. The present disclosure contemplates sortagging of any such
microorganism.
[0479] In some embodiments a sortagged microorganism is contacted
with target cells ex vivo. In some embodiments a sortagged
microorganism is administered to a subject and encounters target
cells in the subject. In some embodiments a subject to whom a
sortagged attenuated microorganism is administered is an individual
who is infected with a non-attenuated strain of the microorganism.
In some embodiments, a sortagged microorganism is administered to a
subject in need of treatment for a tumor. The microorganism may be
sortagged with a targeting moiety that binds to a tumor antigen
expressed by tumor cells in the tumor. In general, the tumor cells
and tumor may be of any type. In some embodiments the tumor is an
ovarian cancer, colon cancer, liver cancer, prostate cancer, lung
cancer, bladder cancer, breast cancer, brain cancer, lymphoma, or
melanoma. In some embodiments a subject to whom a sortagged
microorganism is administered has a tumor composed at least in part
of cells that the microorganism is capable of invading.
[0480] In some embodiments, tumor cells or pathogenic organisms,
e.g., pathogenic eukaryotic organisms, are sortagged with an agent
comprising a binding moiety that binds to a cell surface molecule
expressed by an antigen presenting cell. In some embodiments the
APC is a professional APC, such as a dendritic cell or macrophage.
In general, the tumor cells may be derived from a tumor of any
tumor type. In general, a pathogenic organism may be any organism
capable of causing disease in a mammal, e.g., a human. In some
embodiments the organism is a fungal cell or parasite. In some
embodiments the organism is a microorganism. In some embodiments
the microorganism is T. gondii. Those of ordinary skill in the art
will be aware of various strains and suitable methods of obtaining
and propagating pathogenic organisms. In some embodiments the
binding moiety serves as a targeting moiety to target the tumor
cells or pathogenic organism to APC. In some embodiments the APC is
a phagocytic cell. In some embodiments the cell surface molecule is
a receptor expressed by the APC. In some embodiments the cell
surface molecule is an MHC Class II molecule, CD205 (DEC205),
DNGR-1 (CLEC9A), CD207 (Langerin), CD11c, CD141, CD303, CD103,
CD209 (DC-SIGN), CD68, or CD163. In general, the binding moiety may
be of any of the various types of binding moieties described
herein. In some embodiments the binding moiety comprises a single
chain antibody (e.g., an scFv) or single domain antibody. For
example, in some embodiments the binding moiety comprises a VHH,
e.g., a VHH that binds to human MHC Class II or a VHH that binds to
human DEC205. VHH4 is an exemplary VHH that binds to human MHC
Class II (described in WO/2013/155526). Exemplary monoclonal
antibodies that bind to human DEC205 are described in Park, C J, et
al., J Immunol Methods. 2012; 377(1-2):15-22. In some embodiments
the tumor cells are obtained from a subject suffering from a tumor.
The tumor cells may be obtained by biopsy or surgery to remove at
least a portion of the tumor or in a blood sample or other
biological sample obtained from the subject. In some embodiments
the tumor cells are from a tumor cell line, which may be derived
from tumor cells obtained from a subject in need of treatment for a
tumor or may be derived from a tumor of the same type as that of a
subject. In some embodiments the tumor cells may express at least
one tumor antigen.
[0481] In some embodiments tumor cells or pathogens that have been
sortagged with a binding moiety that binds to a cell surface
molecule expressed by an APC are processed. In some embodiments,
processing renders the tumor cell non-viable, less viable (reduced
lifespan), or incapable of proliferating. In some embodiments the
processing renders the pathogen non-viable, less viable,
attenuated, non-infectious, and/or incapable of proliferating. For
example, the tumor cell or pathogen may be exposed to radiation or
a toxic substance such as a pro-apoptotic agent. In some
embodiments the processing may comprise physical or chemical
fragmentation, lysis, or fractionation. For example, cells may be
sonicated, subjected to bead beating, dounced, sheared, subjected
to conditions of high or low osmolarity sufficient to induce cell
lysis, exposed to a detergent, and/or exposed to a cytolytic agent.
In some embodiments a cell membrane fraction may be isolated from
the sortagged tumor cells or pathogens. In some embodiments, tumor
cells or pathogens may be processed in any of the afore-mentioned
ways prior to sortagging. Without wishing to be bound by any
theory, fragmentation, lysis, or fractionation may produce portions
of cells that are more readily phagocytosed or endocytosed than a
whole cell.
[0482] In some embodiments a tumor cell or pathogen that has been
sortagged with a binding moiety that binds to a cell surface
molecule expressed by an APC is used to deliver an antigen
expressed by the tumor cell or pathogen to APC that express the
cell surface molecule. By binding to the APC, the binding moiety
maintains the sortagged tumor cell or pathogen in close proximity
to the APC, which may then internalize the tumor cell or pathogen
or a portion thereof or a product thereof such as a molecule shed
or released from the tumor cell or pathogen. For example, the APC
may internalize the tumor cell, pathogen, or a portion or product
thereof by phagocytosis or endocytosis. In some embodiments a
pathogen is able to invade the APC and is thereby internalized. The
APC may process the internalized tumor cell, pathogen, or portion
or product thereof and present one or more antigens derived from
the tumor cell, pathogen, or portion or product thereof on its
surface in association with an MHC Class I or Class II molecule.
For example, the APC may present a peptide comprising one or more
epitopes derived from the tumor cell, pathogen, or portion of
product thereof. In some embodiments tumor cells or pathogens that
have been sortagged with a binding moiety that binds to a cell
surface molecule expressed by an APC are administered to a subject,
whereupon the binding moiety binds to APCs in the subject. In some
embodiments tumor cells or pathogens that have been sortagged with
a binding moiety that binds to a cell surface molecule expressed by
an APC may be contacted with APCs ex vivo, e.g., by placing them in
the same vessel, whereupon the binding moiety attached to the tumor
cells or pathogens binds to the APCs. In some embodiments the APCs
may subsequently be administered to a subject or may be contacted
ex vivo with T cells that are subsequently administered to the
subject. The APC may present the antigen to T cells ex vivo or in a
subject. Presentation of the antigen by the APC may elicit or
enhance a T cell response in the subject or ex vivo. The response
may be directed toward cells that express the antigen, such as
tumor cells or pathogens in the subject. An APC may thus elicit or
promote an immune response against a tumor or pathogen in a
subject. In some embodiments an APC may stimulate T cells by
presenting an antigen derived from the sortagged tumor cell or
pathogen to such cells. In some embodiments the T cells are naive T
cells. In some embodiments an APC may stimulate a cytotoxic T cell
response, a helper T cell response, or both.
[0483] In some embodiments a tumor cell or pathogen that has been
sortagged with a binding moiety that binds to a cell surface
molecule expressed by an APC is also sortagged with a second agent.
In some embodiments the second agent is capable of stimulating the
functional maturation or at least one biological activity of an
APC. In some embodiments the second agent comprises an adjuvant. In
some embodiments a tumor cell or pathogen that has been sortagged
with a binding moiety that binds to a cell surface molecule
expressed by an APC is administered in combination with an
adjuvant. The adjuvant may be in the same composition or may be
administered separately.
[0484] In some embodiments a subject to whom sortagged tumor cells
or portions thereof, sortagged pathogens or portions thereof, or
APCs that have been contacted ex vivo with sortagged tumor cells,
pathogens, or portions thereof, are administered is in need of
treatment for a tumor or is in need of treatment for an infection
caused by the pathogen. In some embodiments, the sortagged tumor
cells or tumor cell portions are derived from the subject's tumor
or from a tumor of the same type as that for which the subject
needs treatment. For example, a subject in need of treatment for a
melanoma may be treated with sortagged melanoma cells; a subject in
need of treatment for a colon carcinoma may be treated with
sortagged colon carcinoma cells, etc. In some embodiments tumor
cells or pathogens that have been sortagged with a binding moiety
that binds to a cell surface molecule expressed by an APC are
processed prior to administration to a subject or prior to
contacting them with APCs in vitro. In some embodiments tumor cells
or pathogens are processed, and the resulting portions are
sortagged, before administration to a subject or contacting with
APC in vitro. Processing may comprise physical or chemical
fragmentation, lysis, or fractionation, e.g., as described above.
In some embodiments administration of tumor cells or pathogens or
portions thereof that have been sortagged with a binding moiety
that binds to APC results in accumulation of the tumor cells,
pathogens, or portions thereof, in lymphoid tissue, e.g., in lymph
nodes or other lymphoid organs. The tumor cells, pathogens, or
portions thereof may come in contact with additional APC, or other
immune system cells, in the lymphoid tissue. In some embodiments
one or more doses of tumor cells or portions thereof that have been
sortagged with a binding moiety that binds to a cell surface
molecule expressed by an APC are administered to a subject prior
to, concurrently with, and/or after surgery, radiation, or
chemotherapy for treatment of a tumor.
[0485] In some embodiments, methods described above in which a
eukaryotic organism is sortagged with an agent comprising a binding
moiety that binds to an APC are applied using a eukaryotic
organisms that is not pathogenic but expresses one or more proteins
that is also expressed by a pathogenic eukaryotic organism or
contains one or more epitopes of such a protein. In some
embodiments cells derived from a multicellular pathogenic
eukaryotic organism are used. It should be noted that the tumor
cells or pathogenic eukaryotic organisms may in some embodiments be
genetically engineered to produce one or more molecules, e.g., a
cytokine, targeting moiety, costimulator, antigen, and/or toxic
substance.
[0486] Sortagged tumor cells, microorganisms, pathogens, or
portions thereof, may be prepared under appropriate conditions
(e.g., in compliance with Good Manufacturing Practices) such that
the resulting preparation is suitable for administration to a
mammalian subject, e.g., a human subject. The sortagged tumor
cells, microorganisms, pathogens, or portions thereof, may be mixed
with a pharmaceutically acceptable carrier. Sortagged tumor cells
or portions thereof, sortagged eukaryotic pathogens or portions
thereof, may be administered to a subject using any suitable
administration route. In some embodiments such cells or portions
thereof are administered intravenously, orally, by inhalation, or
topically. In some embodiments such cells or portions thereof are
administered locally, e.g., directly to a tumor site, e.g., by
injection into a tumor or intraperitoneally in the case of a
peritoneal tumor, or locally to a site of infection (e.g., the
lungs, liver, etc.) The number of cells to be admininstered in a
dose and the number of doses to be administered can be determined
using routine procedures known to those of ordinary skill in the
art. In certain embodiments sortagged tumor cells or portions
thereof, sortagged eukaryotic pathogens or portions thereof, are
administered to a subject in combination with sortagged immune
system cells, e.g., sortagged T cells.
[0487] The following working examples are intended to describe
exemplary reductions to practice of certain methods, reagents, and
compositions provided herein and do not limit the scope of the
invention.
EXAMPLES
Example 1: Sortase-Catalyzed Conjugation of Biotin Probe to
Non-Genetically Engineered Mammalian Cells
[0488] Mouse red cell-depleted splenocytes were isolated using
standard methods. Approximately 2 million cells were incubated with
a biotin-LPETG probe (1 mM) either with or without sortase A (150
.mu.M) from Staphylococcus aureus in 100 microliters DME without
serum at 4.degree. C. for 1 hour. Cells were then washed with PBS 5
times and lysed in Laemmli buffer. Samples were run on an SDS-PAGE
gel. Approximately one quarter of the lysate from each incubation
was loaded per lane. Biotinylated proteins were visualized by
blotting with streptavidin-HRP using standard methods. The
resulting immunoblot is shown in FIG. 2. The blot demonstrates
labeling of a number of proteins with the biotin-LPETG probe. Based
on other experiments, the prominent band of about 30 kD is believed
to be sortase itself labeled with biotin-LPETG, while the band at
about 60 kD is likely to be biotin-labeled sortase dimer.
[0489] Flow cytometry analysis was used to confirm
sortase-catalyzed labeling of non-genetically engineered mammalian
cells with the biotin-LPETG probe (1 mM). Mouse red cell-depleted
splenocytes isolated using standard methods were incubated with
biotin-LPETG probe with or without sortase A (200 .mu.M) from
Staphylococcus aureus in DME without serum at 4.degree. C. for 1
hour. Cells were then washed with PBS 5 times, incubated with
phycoerythrin (PE)-conjugated streptavidin, and subjected to flow
cytometry. Results are shown in FIG. 3. Blue histograms (indicated
with arrows) show PE signal gated on living cells incubated with
(right) or without (left) sortase A. Black histograms (no arrow)
show background staining on control splenocytes. The results show
specific binding of PE-conjugated streptavicdin only to the cells
that had been incubated with the biotin-LPETG probe in the presence
of sortase, thus demonstrating the labeling of these cells by
sortase-catalyzed conjugation of the probe thereto.
[0490] The production and purification of sortase used in this
experiment is described in Popp, M W, et al., Nat Chem Biol. 2007;
3(11):707-8. Epub 2007 Sep. 23.
[0491] The biotin-LPETG probe was prepared according to the
following protocol, which also describes preparation of a
TAMRA-LPETG probe. These probes have two Gs at the C-terminus,
i.e., biotin-LPETGG and TAMRA-LPETGG.
A) TAMRA-LPETGG Probe
[0492] Note: Use Fmoc-Ala-OH in place of Fmoc-Gly-OH to make probes
for S. pyogenes sortase A
Resin Preparation Timing 15 Min
[0493] i Add 100 .mu.mol of Rink amide resin (167 mg, 0.6 mmol/g)
into a capped glass column with a fritted glass filter bottom,
solvate the resin in dichloromethane (DCM) (7 mL) by shaking for 15
min in a wrist-action shaker and remove the DCM by vacuum
filtration.
Deprotection Timing 30 Min
[0494] ii Add 20% piperidine solution in N-methyl-2-pyrrolidone
(NMP) (7 mL) and shake for 15 min to remove the resin's Fmoc
protecting groups. Note: In all steps NMP may be replaced with DMF.
iii Remove the piperidine solution by vacuum filtration and wash
the resin three times with NMP (7 mL, 1 min), three times with DCM
(7 mL, 1 min) and an additional time with NMP (1 min).
Coupling Reaction Timing 2-3 h Until Pause Point, 3.5 h Per
Coupling Cycle
[0495] iv Dissolve Fmoc-Gly-OH (89 mg, 300 mol), HBTU (114 mg, 300
mol), and DIPEA (104 .mu.L, 600 .mu.mol) in NMP (7 mL) and add to
the resin. Shake the suspension for 2 h at room temperature. v
Remove the reaction solution by vacuum filtration and wash the
resin three times with NMP (7 mL, 1 min) and three times with DCM
(7 mL, 1 min). The coupling reaction can be confirmed by performing
a Kaiser test. Note: If the reaction is incomplete repeat steps
iv-v with half the amount of reagents used for a standard coupling
and shake for 1 h. PAUSE POINT: The resin can be stored at
4.degree. C. after drying under vacuum. At this stage, store
peptides in their Fmoc-protected form, if storage is desired. vi
Repeat steps i-v with Fmoc-Gly-OH (89 mg, 300 mol),
Fmoc-Thr(OtBu)-OH (119 mg, 300 mol), Fmoc-Glu(OtBu)-OH (127 mg, 300
mol), Fmoc-Pro-OH (101 mg, 300 mol), Fmoc-Leu-OH (106 mg, 300 mol),
Fmoc-c-aminocaproic acid (85 mg, 300 mol). Note: The Kaiser test
does not work for verifying the extent of the Leu coupling, since
the N-terminus of Pro is a secondary amine. To test this coupling
reaction, one can use the chloroanil test or microcleavage. Note
that the orthogonal protecting groups may not be fully removed
during this abbreviated cleavage. vii After removing the Fmoc on
the .epsilon.-aminocaproic acid residue, add a solution of
5(6)-TAMRA (52 mg, 120 mol), PyBOP (63 mg, 120 mol), and DIPEA (42
.mu.L, 240 mol) in NMP (7 mL) and shake overnight at room
temperature. To prevent photobleaching of the fluorophore, wrap the
column in aluminum foil. viii Repeat step v and perform the Kaiser
test to check the TAMRA coupling. Cleavage from Resin ix Suspend
the resin in cleavage solution consisting of 95% TFA, 2.5%
H.sub.2O, and 2.5% TIS (5 mL) for 2 h at room temperature. x Elute
the cleavage solution into 90 mL of ice cold (-0.degree. C.)
diethyl ether and rinse the resin with an additional 3 mL of the
cleavage solution into the ether. xi Store the ether solution at
-20.degree. C. for 20 min to precipitate the peptide. Centrifuge
the suspension at 1,900 g for 15 min at 4.degree. C., decant the
supernatant and gently evaporate the remaining ether under reduced
pressure. Due to the flammable and volatile nature of diethyl ether
it is desirable to use a spark-free freezer and centrifuge. Pause
point: The crude peptide can be stored as a solid at -20.degree. C.
Critical step: The identity and purity can be verified by LC/MS
analysis (linear gradient 5.fwdarw.45% LC/MS buffer B over 10 min).
If LC/MS shows that the crude peptide is of sufficient purity, the
next steps (xii-xiv) may be omitted and the peptide may be used
directly in sortase reactions.
HPLC Purification
[0496] xii Dissolve the dried peptide in H.sub.2O (2 mL) and
centrifuge at 14,000 rpm for 10 min in a tabletop centrifuge to
remove particulate matter. Note: Up to 50% of tert-butanol may be
added to peptides that do not dissolve in pure H.sub.2O. Also spin
filters or syringe filters may be used to remove particulate
matter. xiii Purify the centrifuged supernatant by reverse-phase
HPLC on a C18 column using a 10-70% buffer B gradient over 15 min,
followed by flushing at 90% buffer B for 5 min. We recommend a
preliminary small 100 .mu.L injection and adjusting the gradient
accordingly for peptide purity and ease of separation. Once a good
gradient is established, the remaining crude material may be
purified with 400-600 .mu.L injections. xiv Analyze the fractions
for product by LC/MS and lyophilize the desired fractions to
dryness. Note: TAMRA containing probes consist of a mixture of
regio-isomers that will likely result in two product peaks during
reverse phase HPLC purification. The different isomers have no
effect on labeling. The identity and purity can be verified by
LC/MS analysis (linear gradient 5.fwdarw.45% LC/MS buffer B over 10
min) and NMR spectroscopy. Pause point: The lyophilized peptide can
be stored at -20.degree. C. indefinitely.
B) Biotin-LPETGG Probe
[0497] i Use the same reaction conditions as for synthesis of the
TAMRA-LPETGG probe through the Fmoc deprotection step of the Leu
residue. At this point, add a solution of biotin (74 mg, 300
.mu.mol), HBTU (114 mg, 300 mol) and DIPEA (104 .mu.L, 600 mol) in
NMP (7 mL); shake for 2 h. ii Remove the reaction solution by
vacuum filtration, wash the resin, and check the success of biotin
coupling with a Kaiser test (remaining free amines). iii Cleave the
product from the resin as indicated in steps ix-xi for the TAMRA
probe. iv Purify by reverse phase HPLC as indicated in steps
xii-xiv of the TAMRA probe The identity and purity can be verified
by LC/MS analysis (linear gradient 5.fwdarw.45% B in 10 min) and
NMR spectroscopy. Pause point: The lyophilized peptide can be
stored at -20.degree. C. indefinitely.
Example 2: Sortase-Catalyzed Conjugation of VHH Protein to
Non-Genetically Engineered Mammalian Cells
[0498] Approximately 2 million mouse red cell-depleted splenocytes
isolated using standard methods were incubated with a GFP-specific
VHH that contains a C-terminal LPETG (100 .mu.M) (see Kirchhofer,
A., et al., Nat Struct Mol Biol. 2010 January; 17(1):133-8. doi:
10.1038/nsmb.1727. Epub 2009 Dec. 13 for description of the
original GFP-specific VHH, which does not contain a C-terminal
LPETG), either with or without sortase A (200 .mu.M) from
Staphylococcus aureus, in DME without serum at 4.degree. C. for 1
hour. Cells were then washed with PBS 5 times, incubated with GFP
for 30 minutes and subjected to flow cytometry. Results are shown
in FIG. 4. Blue histograms show GFP signal gated on living cells
that had been incubated with (right) or without (left) sortase A.
Black histograms show background staining on control splenocytes
incubated with GFP. The blue and black histograms in the left panel
are virtually superimposable. The blue histogram in the right panel
is indicated with an arrow. The results show specific binding of
GFP only to the cells that had been incubated with the GFP-specific
VHH in the presence of sortase, demonstrating the labeling of these
cells by sortase-catalyzed conjugation of the VHH thereto.
Example 3: Sortase-Catalyzed Conjugation of VHH Protein to
Non-Genetically Engineered Mammalian Cells
[0499] Lymphocytes are isolated from mice using standard methods,
and expanded and activated in vitro using appropriate antibodies
(e.g., soluble or immobilized anti-CD3 mAb) and/or cytokines (e.g.,
IL-2) for T cell expansion and activation. An aliquot of the
expanded and activated cells is incubated in culture medium with
either a human tumor antigen (TA)-specific VHH (anti-TA VHH)
containing a C-terminal LPETG or an anti-GFP VHH containing a
C-terminal LPETG, either with or without sortase A from
Staphylococcus aureus at 4.degree. C. for 1 hour. Cells are then
washed with PBS 5 times, incubated with a recombinant tumor antigen
labeled with fluorescein isothiocyanate (FITC) fluorescent dye, and
subjected to flow cytometry. Staining of the cells for FITC is
compared with staining of control cells that had been incubated
with the labeled recombinant tumor antigen but not with the VHH.
Increased FITC signal from the cells that had been incubated with
tumor-antigen specific VHH and sortase, followed by incubation with
labeled tumor antigen, as compared with staining of control
lymphocytes either (i) incubated with tumor-antigen specific VHH in
the absence of sortase followed by followed by incubation with
labeled tumor antigen; or (ii) incubated with anti-GFP VHH in the
presence of sortase followed by incubaton with labeled tumor
antigen indicates successful sortase-mediated conjugation of the
tumor-antigen specific VHH to the cells.
[0500] Cytotoxic activity of lymphocytes labeled with either
anti-TA VHH or anti-GFP VHH towards target cells expressing the
tumor antigen at their surface is assessed in vitro using standard
methods such as chromium release assays and compared with cytotoxic
activity of control lymphocyes.
[0501] Lymphocytes labeled with either anti-TA VHH or anti-GFP VHH
are administered to separate groups of mice bearing xenografts of
human tumor cells expressing the tumor antigen at their surface.
Tumors are isolated after 2-6 weeks and their size and weight
determined and compared among groups.
Example 4: Sortase-Catalyzed Conjugation of VHH Protein to
Non-Genetically Engineered Mammalian Cells
[0502] Lymphocytes are isolated from mice using standard methods,
expanded and activated in vitro using appropriate antibodies (e.g.,
soluble or immobilized anti-CD3 mAb) and/or cytokines (e.g., IL-2)
for T cell expansion and activation, and incubated in culture
medium with a human tumor antigen-specific VHH containing a
C-terminal HA-LPETG or a GFP-specific VHH containing a C-terminal
HA-LPETG, either with or without sortase A from Staphylococcus
aureus at 4.degree. C. for 1 hour. Cells are then washed with PBS 5
times, incubated with anti-HA antibody labeled with fluorescein
isothiocyanate (FITC) fluorescent dye, and subjected to flow
cytometry. Staining of the cells for FITC is compared with staining
of control cells that had been incubated with the labeled
recombinant tumor antigen but not with the VHH. Increased FITC
signal from the cells that had been incubated with tumor-antigen
specific VHH and sortase followed by incubation with labeled tumor
antigen, as compared with background staining of control cells
incubated with tumor-antigen specific VHH in the absence of sortase
followed by followed by incubation with labeled tumor antigen
indicates successful sortase-mediated conjugation of the
tumor-antigen specific VHH to the cells.
[0503] Cytotoxic activity of lymphocytes with either HA-tagged
anti-TA VHH or HA-tagged anti-GFP VHH conjugated thereto towards
target cells expressing the tumor antigen at their surface is
assessed in vitro using standard methods such as chromium release
assays and compared.
[0504] Lymphocytes with HA-tagged anti-TA VHH or HA-tagged anti-GFP
VHH conjugated thereto are administered to mice bearing xenografts
of human tumor cells expressing the tumor antigen. Tumors are
harvested 2 hours later and analyzed for presence of administered
lymphocytes using immunohistochemistry, staining with antibodies
against either the HA tag, CD3 (to detect T cells), or both.
Increased number of T cells in the tumors of mice to which the
lymphocytes sortagged with HA-tagged anti-TA VHH were administered
as compared with the number of T cells in the tumors of mice to
which lymphocytes sortagged with HA-tagged anti-GFP VHH were
administered demonstrates tumor targeting of the lymphocytes
[0505] In another experiment lymphocytes with either HA-tagged
anti-TA VHH or HA-tagged anti-GFP VHH conjugated thereto are
administered to separate groups of immunocompromised mice bearing
xenografts of human tumor cells expressing the tumor antigen at
their surface. Tumors are isolated after 2-6 weeks and their
average and total size and weight are determined and compared among
groups. Reduced average and/or total tumor size and weight in the
mice to which the lymphocytes sortagged with the anti-TA VHH were
administered as compared with the mice to which the lymphocytes
sortagged with anti-GFP VHH were administered is indicative that
sortagging with a tumor-targeting moiety can improve efficacy of
adoptive anti-tumor immunotherapy.
Example 5: Sortase-Catalyzed Conjugation of VHH Protein to
Non-Genetically Engineered Human Lymphocytes
[0506] Peripheral blood mononuclear cells (PBMC) from a human donor
are isolated using standard methods, expanded and activated in
vitro using appropriate antibodies (e.g., soluble or immobilized
anti-CD3 mAb) and/or cytokines (e.g., IL-2) for T cell expansion
and activation. An aliquot of the expanded and activated cells is
incubated in culture medium with a human tumor antigen-specific VHH
containing a C-terminal LPETG or an anti-GFP VHH containing a
C-terminal LPETG, either with or without sortase A from
Staphylococcus aureus at 4.degree. C. for 1 hour. Cells are then
washed with PBS 5 times, incubated with a recombinant tumor antigen
labeled with either fluorescein isothiocyanate (FITC) or Cy7.5
near-infrared fluorescent dye, and subjected to flow cytometry.
Staining of the cells for FITC or Cy7.5 is compared with staining
of control cells that had been incubated with the labeled
recombinant tumor antigen but not with the VHH. Increased FITC or
Cy7.5 signal from the cells that had been incubated with
tumor-antigen specific VHH and sortase followed by incubation with
labeled tumor antigen, as compared with background staining of
control cells incubated with tumor-antigen specific VHH in the
absence of sortase followed by followed by incubation with labeled
tumor antigen indicates successful sortase-mediated conjugation of
the tumor-antigen specific VHH to the cells.
[0507] Cytotoxic activity of lymphocytes labeled with either human
anti-TA VHH or anti-GFP VHH towards target cells (e.g., human tumor
cells) expressing the tumor antigen at their surface is assessed in
vitro using standard methods such as chromium release assays.
[0508] Lymphocytes labeled with either human anti-TA VHH or
anti-GFP VHH are administered to immunocompromised mice bearing
human tumor xenografts that express the TA at their surface. Tumors
are isolated after 2-6 weeks and their average and total size and
weight are determined and compared among groups. Reduced average
and/or total tumor size and weight in the mice to which the
lymphocytes sortagged with the anti-TA VHH were administered as
compared with the mice to which the lymphocytes sortagged with
anti-GFP VHH were administered is indicative that sortagging with a
tumor-targeting moiety can improve efficacy of adoptive anti-tumor
immunotherapy.
Example 6: Sortase-Catalyzed Conjugation of Antibodies to
Non-Genetically Engineered Mammalian Lymphocytes
[0509] Examples 3-5 are repeated except that a conventional human
antibody comprising a chain that contains a sortase recognition
sequence is used instead of a VHH.
Example 7: Sortase-Catalyzed Conjugation of Antibodies to
Non-Genetically Engineered Mammalian Lymphocytes
[0510] Examples 3-5 are repeated except that an scFv comprising a
sortase recognition sequence is used instead of a VHH.
Example 8: Sortase-Catalyzed Conjugation of Antibodies to
Non-Genetically Engineered Mammalian Lymphocytes
[0511] Examples 3-7 are repeated with the additional step(s) of (i)
enriching for CD8+ cells using MACS beads prior to sortagging
(using, e.g., CD8+ T Cell Isolation Kit, human (#130-096-495),
Miltenyi Biotec); (ii) applying co-stimulation using an antibody to
CD28 prior to administration of the sortagged lymphocytes; and/or
(iii) assessing cytotoxic activity of a sample of the sortagged
lymphocytes in vitro by measuring secretion of granzyme and/or
perforin and/or by assessing CD107 cell surface expression using
anti-CD107 mAbs.
Example 9: Sortase-Catalyzed Conjugation of Sortase Substrate to
Genetically Unmanipulated Eukarotyic Cells of Diverse Species
[0512] Antibodies used in Examples 9, 10, 11, and/or 28: Anti-PGK
(clone 22C.sub.5D8, Invitrogen), anti-mouse/human actin (clone
Ab-5, BD biosciences), anti-Toxoplasma gondii actin, horseradish
peroxidase-conjugated goat anti-rabbit Ig (Southern Biotech, cat.
number 4041-05), horseradish peroxidase-conjugated anti-mouse Ig
(GE Healthcare, cat. number NXA931), anti-TCRbeta (clone H57, BD
Pharmingen), anti-CD4 (clone GK1.5, ebiosciences), anti-CD19 (clone
1D4, BD Pharmingen), anti-TER119 (clone TER-119, BD Pharmingen),
allophycocyanin-conjugated streptavidin (ebiosciences, cat. number
17-4317), phycoerythrin-conjugated streptavidin (Southern Biotech,
cat. number 7100-09S). Propidium iodide (Sigma-Aldrich, cat. number
P4864).
[0513] Results:
[0514] Saccharomyces cerevisiae (W303), Toxoplasma gondii, HEK 293
T cells, or total mouse splenocytes from WT C57BL/6 mice were
incubated 1 hour at room temperature with or without 500 .mu.M of
biotin-LPETG and with or without 20 .mu.M of a Ca2+-independent
sortase A (see description in Example 11). HEK 293T cells were
incubated at 20 million per milliliter, Toxoplasma gondii at 20 to
40 million per milliliter, and yeast at 6 OD 280 units per
milliliter. Conjugation of biotin-LPETG probes was analyzed by SDS
page followed by Western blotting using Streptavidin HRP. Results
are presented in FIGS. 5(A)-(D). The right lane of each blot
contains a number of bands clearly showing the sortase-catalyzed
labeling of multiple proteins in each cell type.
[0515] Total mouse splenocytes (20-100 million cells per
milliliter) from WT C57BL/6 mice were incubated 1 hour at room
temperature with 500 .mu.M of biotin-LPETG and with (dark grey
histograms) or without (light grey histograms) 20 .mu.M of sortase
A. Conjugation of biotin-LPETG was analyzed by flow cytometry using
fluorescently labeled streptavidin together with antibody specific
for T (TCRb), B (CD19), or red cells (Ter119). Results are
presented in FIG. 5(E) and clearly show the presence of labeled
cells of each cell type. Biotin-LPETG probes labeled T and B cells
equally well, and slightly less efficiently, red cells.
[0516] We measured by flow cytometry the kinetics with which
biotin-LPETG was conjugated to red cell-depleted splenocytes by
flow cytometry. Conjugation reached .about.30% of maximum after 5
minutes and .about.60% of maximum after 15 minutes (FIG. 5(F)).
Collectively, our data show that all cells tested were efficiently
sortagged in a time frame compatible with the investigation of many
biological processes. Presumably the vast majority of cells have
naturally exposed glycines at their cell surface and will therefore
be amenable to direct sortagging.
Example 10: Measurement of Immune System Cell-Mediated Cytotoxicity
Towards Specific Target Cells
[0517] This example demonstrates that red cell-depleted splenocytes
from OTI Rag.sup.-/- mice contain a cell population capable of
exerting cell-mediated toxicity towards appropriate target cells
upon stimulation and presents a method of quantifying the cytotoxic
effect. Splenocytes are a mixed population of immune system cells
containing a variety of cell populations such as lymphocytes, NK
cells, and macrophages. OTI Rag.sup.-/- mice are deficient for Rag
and are transgenic for a T cell receptor that recognizes the
SIINFEKL peptide in the context of H2.sup.b. These mice produce
CD8.sup.+ cells that are specific for SIINFEKL. Due to the
Rag-deficient status of the mice, they lack B cells and CD4.sup.+ T
cells.
[0518] Splenocytes were isolated from OTI Rag.sup.-/- mice and
depleted of splenocytes by osmotic shock using standard methods.
Red cell-depleted splenocytes from OTI Rag.sup.-/- mice were
incubated in complete RPMI (RPMI 1640 supplemented with 10%
(vol/vol) inactivated FCS, .beta.-mercaptoethanol, non essential
amino acids, sodium pyruvate and penicillin--streptomycin) in a 24
well plate coated with anti-CD3 and anti-CD28 antibody (2 .mu.g/ml
in PBS, 30 min at 37.degree. C.) to stimulate the T cell
population. After 72 hours, cells were mixed with red-cell depleted
C57BL/6 splenocytes (isogenic with the OTI Rag.sup.-/- mice but for
the Rag mutations) that had been pre-incubated for 30 min at
37.degree. C. in complete RPMI plus DMSO or plus DMSO+SIINFEKL
peptide (final concentration 1 .mu.g/ml). The purpose of the
pre-incubation in the presence of SIINFEKL was to load cells within
the C.sub.57BL/6 splenocyte population with SIINFEKL, causing it to
be displayed at the cell surface. The cells were mixed in 96 U
bottom well plates at a 1:1 ratio (200,000 each) in 200 ml complete
RPMI. After 24 hours, B cell viability was measured by staining the
cell population with antibody to CD19 (a marker expressed on B
cells) and propidium iodide (PI) and subjecting the cells to flow
cytometry. Cells that are negative for PI staining are viable.
[0519] FIG. 6 shows dot plots showing viable C57BL/6 B cells
(CD19+, PI-) (indicated in the boxed regions remaining after
incubation of SIINFEKL-loaded (left panel) or control (right panel,
not SIINFEKL-loaded) C57BL/6 splenocytes with the splenocytes from
OTI Rag-/- mice (containing CD8+ cells specific for SIINFEKL). In
the absence of SIINFEKL loading, about 7.5% of the C57BL/6
splenocytes detected (including both viable and non-viable cells)
were viable B cells, while with SIINFEKL loading only about 1.1% of
the C57BL/6 splenocytes were viable B cells. SIINFKEFL loading thus
resulted in a dramatic reduction in the proportion of C57BL/6
splenocytes that were viable B cells following incubation with
splenocytes from OTI Rag-/-mice (1.1% with SIINFEKL loading (right
panel) versus 7.5% without SIINFEKL loading (left panel). This
experiment confirms that splenocytes from OTI Rag-/-mice contain a
population of cells that have the capacity to exert cell-mediated
cytotoxicity towards target cells that bear a specific target
antigen (in this case the peptide SIINFEKL) on their surface and
that the approach described here is suitable to measure
cell-mediated cytotoxicity towards target cells of interest.
Example 11: Non-Genetically Engineered Immune System Cells
Sortagged with a Targeting Moiety Exhibit Cytotoxicity Specific for
Target Cells
[0520] This example demonstrates that attaching a targeting moiety
to non-genetically engineered immune system cells using sortase
increases their cytotoxicity specifically towards cells bearing a
target to which the targeting moiety binds. As in Example 10, the
experiment described in this example makes use of red cell-depleted
splenocytes from OTI Rag.sup.-/- mice. The splenocytes were
sortagged with a VHH (VHH7) that binds to mouse MHC Class I, and
the ability of these cells to exert cytotoxic effects towards
murine B cells expressing MHC Class I was assessed using a similar
approach to that described in Example 9.
[0521] Red cell-depleted splenocytes from OTI Rag.sup.-/- mice were
incubated in complete RPMI in a 24 well plate coated with anti-CD3
and anti-CD28 antibody as described in Example 10. After 72 hours
the cells were washed and incubated with or without Enhancer VHH or
VHH7 (500 .mu.M, 50 .mu.M, or 5 .mu.M) with or without sortase A
(final concentration 20 .mu.M mutated S. aureus srtA, Ca 2+
independent), in a total volume of 200 .mu.l of PBS,
5.times.10.sup.6 cell per reaction at room temperature for 1 hour,
in order to conjugate the relevant VHH to the cell surface. The
Ca2+ independent sortase that was used in this experiment is a
6.times.His tagged version with the following sequence:
TABLE-US-00009 (SEQ ID NO: 7)
MQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATREQLNRGVSFAKENQ
SLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSI
RNVKPTAVEVLDEQKGKDKQLTLITCDDYNEETGVWETRKIFVATEVKLE HHHHHH
[0522] Enhancer VHH refers to a VHH that binds to GFP and has been
modified to contain an LPETG sequence at its C-terminus, thus
permitting the VHH to serve as a sortase substrate (see also
Example 2). As noted above, VHH7 is a VHH that binds to murine MHC
Class II molecules (PCT/US2013/036630 (WO/2013/155526; Witte, M D
et al. (2012) PNAS, 109(30): 11993-11998). A version of VHH7 that
contains a C-terminal LPETG was used in this experiment, thus
permitting the VHH to serve as a sortase substrate. Where the terms
"Enhancer VHH" and "VHH7" are used below and in FIG. 7 (which
presents data from this Example), it should be assumed that the
versions containing a C-terminal LPETG sequence were used.
[0523] Following incubation with VHH7 or Enhancer VHH (or following
control incubation without VHH), in each case either with or
without sortase, red cell-depleted splenocytes from OTI Rag.sup.-/-
mice were incubated with various concentrations of purified GFP
protein or without GFP (Control). Binding of GFP through conjugated
Enhancer-LPETG was analyzed by flow cytometry. As shown in FIG.
7(A), GFP binds to cells that were exposed to Enhancer in the
presence of sortase (lower left panel) but does not bind to cells
that were exposed to Enhancer in the absence of sortase or to cells
that were exposed to VHH7 in the absence or presence of sortase
(other 3 panels). To further demonstrate the sortase-catalyzed
conjugation of VHHs to the cell surface, the amount of GFP bound to
cells that had been incubated under each condition (i.e., in the
absence or presence of sortase, in each case with either Enhancer
or VHH7) was estimated by analyzing cell lysates by SDS-PAGE and
Western blotting against GFP protein and comparing signal to a GFP
standard (right lanes of FIG. 7(B)). As shown in FIG. 7(B), GFP
binds only to the cells that were incubated with Enhancer in the
presence of sortase. The number of VHHs installed was approximately
proportional to the concentration of VHHs used for the
reaction.
[0524] We estimated the number of VHHs installed per cell by
measuring the number of bound GFP molecules by SDSPAGE and
immunoblotting, using a solution of GFP of known concentration as
standard (FIG. 7(B)). Sortagging of T cells in the presence of 500
.mu.M of VHHs and sortase A resulted in the conjugation of .about.1
million VHHs per cell.
[0525] Following incubation with VHH7 or Enhancer VHH (or following
control incubation without VHH), in each case either with or
without sortase, red cell-depleted splenocytes from OTI Rag.sup.-/-
mice were washed and then incubated with red cell-depleted wild
type C57BL/6 splenocytes for 24 h in U bottom 96 well plates at a
1:1 ratio (200,000 each) in 200 .mu.l complete RPMI. After 24
hours, B cell viability among the red cell-depleted wild type
C57BL/6 splenocytes was measured by staining with CD19 antibody and
propidium iodide (PI) and subjecting the cells to flow cytometry.
As a control, viability of CD4.sup.+ T cells among the red
cell-depleted wild type splenocytes was measured by staining with
CD4 antibody and propidium iodide (PI).
[0526] FIGS. 7(C) and 7(D) present bar graphs showing the
percentage of viable C57BL/6 T cells (CD4+) and B cells
(CD19.sup.+, PI.sup.-) in a representative experiment conducted
with VHH7 either in the absence of sortase (left panel) or in the
presence of sortase (right panel). The data indicate that
non-sortagged splenocytes from OTI Rag.sup.-/- mice are not
cytotoxic towards either C57BL/6 B cells or T cells i.e.,
approximately 100% of the C57BL/6 B cells and approximately 100% of
the C57BL/6 T cells remain viable following incubation with
non-sortagged splenocytes from OTI Rag.sup.-/- mice (regardless of
whether such non-sortagged splenocytes from OTI Rag.sup.-/- mice
had been incubated with VHH7, enhancer VHH, or no VHH). The data
presented in FIG. 7(C) indicate that splenocytes from OTI
Rag.sup.-/- mice that have been incubated with sortase alone
(control) are not cytotoxic towards either C57BL/6 B cells or T
cells i.e., approximately 100% of the C57BL/6 B cells and
approximately 100% of the C57BL/6 T cells remain viable following
incubation with splenocytes from OTI Rag.sup.-/- mice that had been
incubated with sortase. The data presented in FIG. 7(D) indicate
that splenocytes from OTI Rag.sup.-/- mice that have been incubated
with sortase and Enhancer VHH are not cytotoxic towards either
C57BL/6 B cells or T cells i.e., approximately 100% of the C57BL/6
B cells and approximately 100% of the C57BL/6 T cells remain viable
following incubation with splenocytes from OTI Rag.sup.-/- mice
that had been incubated with sortase and Enhancer VHH. The data
presented in FIG. 7(D) also indicate that splenocytes from OTI
Rag.sup.-/- mice that have been incubated with sortase and VHH7
(500 micromolar or 50 micromolar) are cytotoxic towards C57BL/6 B
cells, but not towards C57BL/6 T cells. Thus, approximately 100% of
the C57BL/6 T cells remain viable following incubation with
splenocytes from OTI Rag.sup.-/- mice that had been incubated with
sortase and Enhancer VHH, whereas only about 40% or 60% of the
C57BL/6 B cells remain viable following incubation with splenocytes
from OTI Rag.sup.-/- mice that had been incubated with sortase and
500 micromolar or 50 micromolar VHH7, respectively. These results
demonstrate that non-genetically engineered cytotoxic immune system
cells can be conjugated to a targeting moiety (in this case VHH7)
using sortase and, as a result of such conjugation, exert cytotoxic
effects specifically towards target cells that express the specific
target of the binding moiety (in this case MHC Class II) at their
cell surface.
Example 11A: Installation of Two Different Agents on Cells Using
Sortase
[0527] To investigate whether two different probes could be
installed on lymphocytes, we incubated erythrocte-depleted
splenocytes with or without enhancer-LPETG for 60 minutes in the
presence of sortase A, followed by the addition of biotin-LPETG to
the reaction for 15 minutes (FIG. 11). Cells incubated with
enhancer-LPETG prior to biotin-LPETG had similar amounts of
surface-conjugated biotin compared to cells incubated with
biotin-LPTEG alone (FIG. 11). These data suggest that sortagging of
VHHs to cells only minimally affects subsequent conjugation of
biotin-LPETG. It is possible that the smaller LPETG-tagged probes
may have more ready access to surface-displayed nucleophiles left
unoccupied by larger LPETG-tagged proteins.
Example 12: Sortase Conjugation of CAR-Modified T Cells with
Anti-PD-L1 Agent
[0528] Human T cells obtained from a patient with B-cell chronic
lymphoblastic leukemia are genetically modified to express a
chimeric antigen receptor (anti-CD20 single chain Fv region fused
to the transmembrane and intracellular domain of CD3-zeta,
containing signaling domains of CD3-zeta and CD28). The cells are
expanded for 10 days in culture with anti-CD3/anti-CD28 beads, then
incubated with sortase and an agent comprising (i) the
extracellular domain of PD-1 and (ii) a sortase recognition
sequence, and transferred back into the patient. The effect of the
administered cells on the leukemia is monitored.
Example 13: Sortase Conjugation of CAR-Modified T Cells with
Anti-PD-1 Agent
[0529] Human T cells obtained from a patient with B-cell chronic
lymphoblastic leukemia are genetically modified to express a
chimeric antigen cell receptor (anti-CD20 single chain Fv region
fused to the transmembrane and intracellular domain of CD3-zeta,
containing signaling domains of CD3-zeta and CD28). The cells are
expanded for 10 days in culture with anti-CD3/anti-CD28 beads, then
incubated with sortase and an agent comprising an scFv that binds
to PD-1 that has been modified to comprise a sortase recognition
sequence, and are then transferred into the patient. The effect of
the administered cells on the leukemia is monitored.
Example 14: Treatment of Multiple Myeloma with Anti-CS1 or
Anti-BCMA Conjugated NK Cells
[0530] NK cells are isolated from a myeloma patient and expanded in
culture. The cells are incubated with sortase and an anti-CS-1 or
anti-BCMA antibody that has been modified to comprise a sortase
recognition sequence, and are then transferred into the patient.
The treatment is repeated weekly for 12 weeks. The effect of the
administered cells on the multiple myeloma is monitored.
Example 15: Prevention of Metastasis with TRAIL-ES Conjugated
PMBCs
[0531] PBMCs from a patient recently diagnosed with stage III colon
carcinoma are isolated. The cells are incubated with sortase and an
anti-CS-1 or anti-BCMA antibody that has been modified to comprise
a sortase recognition sequence, and are then transferred into the
patient. The treatment is repeated weekly for 12 weeks following
colonic resection. The patient is monitored for presence of local
recurrence or metastasis.
Example 16: Inhibition of Mesothelioma Tumor Growth in a Mouse
Model by Mesothelin-Targeted T Cells
[0532] A modified version of SS1 Fv, an Fv specific for mesothelin,
is generated. The modified Fv has a sortase recognition sequence at
the C-terminus. Human T cells (not genetically modified) are
incubated with sortase and the modified SS1 Fv.
[0533] To assess the therapeutic effect of the SS1Fv-conjugated
cells against established mesothelioma, mesothelioma tumors are
established by intraperitoneal (i.p.) injection of 5 million
LMB-H226-GL cells in 200 .mu.l of growth media into the low abdomen
or flank area of eight week old female athymic nude mice (ATHYMIC
NCr-nu/nu) as described (Feng M, et al., J Cancer 2: 123-131).
LMB-H226-GL cells are a human mesothelioma cell line LMB-H226-GL
generated by Feng, et al by fluorescently labeling the NCI-H226
human mesothelioma cell line by a lentiviral vector harboring a
luciferase-GFP (Luc/GFP) fusion gene driven by the RNA polymerase
II promoter. After single-cell cloning by flow cytometry, a clone
(named LMB-H226-GL) that stably expresses high levels of Luc/GFP
was obtained. The labeled cells can be imaged in vivo, e.g., to
monitor tumor growth. Following introduction of the mesothelioma
cells, animals are imaged the following day and then once every
week thereafter. The animals are divided into 5 groups; (1)
Vehicle, (2) SS1P (0.4 mg/kg), (3) mesothelin-targeted T cells, (4)
IL12-SS1 (Fv) (1.6 mg/kg body weight); (5) IL12-SS1 (Fv) (1.6 mg/kg
body weight)+ cells. IL12-SS1 (Fv) is a recombinant immunocytokine
in which IL12 is fused to the anti-mesothelin antibody scFv (SS1).
The p40 and p35 subunits of murine IL12 are connected by flexible
linker (Ser4Gly)3. IL12-SS1 is described further in Kim, H., et
al., PLoS One. 2013 Nov. 15; 8(11):e81919. doi:
10.1371/journal.pone.00819190.
[0534] The tumor-bearing mice are treated with the respective
treatments or vehicle every the other day. The day when the mice
were injected with the tumor cells is considered as day 1. SS1P,
anti-MSLN scFv conjugated with Pseudomonas toxin, is used as a
positive control. The treatment groups and control group each
contain 5 mice. Each mouse in the treatment groups receives 0.4
mg/kg body weight of SS1P, 0.4 mg/kg (low dose group), or 1.6 mg/kg
(high dose group) of IL12-SS1 (Fv) every other day. The control
group receives PBS as a vehicle control. Body weight and tumor
growth are assessed twice a week.
[0535] Assessment of tumor growth is performed as follows: Two
hundred microliters of 15 mg/mL D-luciferin (Caliper Life Sciences,
Hanover, Md.) in PBS are injected i.p. before imaging. The
luciferase activity of the tumor os calculated using Living Image
3.1.0 software (Caliper Life Sciences, Hanover, Md.).
Intraperitoneal Tumor growth was assessed using photon intensity,
photons per second (ph/sec) as luciferase activity as described
(Feng, et al, cited earlier in this example). At the end of the
treatment, three mice in each group are euthanized. Blood is taken
for whole blood complete blood counts (CBC) and serum chemistry
analysis. A full necropsy is performed, in which organs and tissues
are weighed and examined for gross findings. Statistical analysis
is performed with Prism (version 5) for Windows (GraphPad Software,
La Jolla, Calif.). Raw data are analyzed by "analysis of variance"
with Dunnett's and Newman-Keuls multiple comparison post tests. p
values<0.05 are considered statistically significant.
Example 17: Treatment of Ovarian Cancer in a Mouse Model with
Anti-CA125 Antibody Conjugated NK Cells
[0536] Human NK cells are incubated with sortase and an anti-CA-125
antibody that has been modified to comprise a sortase recognition
sequence. Following conjugation, NK cells are cultured with OVCAR
cells (an epithelial ovarian cancer cell (EOC) line that expresses
CA125 and mesothelin). Cells are stained with propidium iodide and
analyzed by FACS. The ability of the CA-125-targeted NK cells to
kill OVCAR cells in vitro is confirmed.
[0537] Efficacy of CA-125-targeted NK cell therapy in vivo either
as single agent or in combination with docetaxel (DTX) (a standard
chemotherapeutic agent used to treat ovarian cancer) is assessed in
an intraperitoneal (i.p.) EOC mouse model that utilizes a subline
of the OVCAR cell line termed OVCAR-3. (Further details of the
OVCAR3 cells and model are described in Pourgholami M H, et al.
Clin Cancer Res 12: 1928-1935 and Wang L, et al. PLoS ONE 6(9):
e24405. doi:10.1371/journal.pone.0024405.). OVCAR-3 cells are
implanted intraperitoneally in female athymic nude mice and allowed
to grow tumor and ascites. Mice are then treated with various
number of CA-125-targeted NK cells, various concentrations of DTX,
combination test (CA-125-targteted NK cells and DTX), combination
control (unconjugated human NK cells and DTX) or vehicle control
i.p for 3 weeks. Treated mice are killed 4 weeks post-treatment.
Ascites volume, tumor weight, CA125 levels from ascites, and
survival of animals are assessed. The expression of MUC1 (tumor
antigen expressed by OVCAR cells), CD31, Ki-67 (proliferation
marker), TUNEL and apoptotic proteins in tumor xenografts was
evaluated by immunohistochemistry. The ability of CA-125-targeted
NK cells to inhibit i.p. tumor development, growth, and ascites
production in a dose-dependent manner is assessed.
Example 18: Treatment of Ovarian Cancer in a Mouse Model with
Anti-CA125 Antibody Conjugated T Cells
[0538] The preceding example is repeated using human T cells
obtained from the patient and expanded in culture.
Example 19: Treatment of Ovarian Cancer with Anti-CA125 and
Anti-Mesothelin Antibody Conjugated NK Cells
[0539] NK cells are isolated from a patient with stage III primary
ovarian cancer and expanded in culture. An aliquot of the cells are
incubated with sortase and an anti-CA-125 antibody that has been
modified to comprise a sortase recognition sequence. A second
aliquot of the cells is incubated with sortase and an
anti-mesothelin antibody that has been modified to comprise a
sortase recognition sequence.
[0540] Cells from each sortagged population are transferred back
into the patient. The treatment is repeated weekly for 12 weeks
following surgery. The effect of the administered cells on residual
cancer and metastases is monitored. The patient is monitored for
blood levels of CA-125 and is retreated with CA-125-targeted NK
cells if an elevated CA-125 level is detected.
Example 20: Treatment of Ovarian Cancer with Anti-CA125 and
Anti-Mesothelin Antibody Conjugated PBMCs
[0541] The preceding example is repeated except that PBMCs obtained
from the patient are used.
Example 21: Treatment of Ovarian Cancer with Anti-CA125 and
Anti-Mesothelin Antibody Conjugated Allogeneic NK Cells
[0542] The preceding example is repeated except that allogeneic NK
cells, in this case NK-92 cells, are used
Example 22: Treatment of Ovarian Cancer with IL-12-Conjugated CAR T
Cells
[0543] Human T cells obtained from a patient with stage III ovarian
cancer are genetically modified to express a chimeric T cell
receptor (anti-CA125 single chain Fv region fused to the
transmembrane and intracellular domain of TCR, containing signaling
domains of CD3-epsilon and CD28). The cells are expanded for 10
days in culture with CD3-CD28 beads and then incubated with sortase
and a fusion protein that contains the two subunits of human
interleukin-12 as a single polypeptide with a sortase recognition
sequence at the C-terminus, and transferred back into the patient
after surgery. Treatment is repeated weekly for 12 weeks. The
effect of the administered cells on residual cancer and metastases
is monitored. The patient is monitored for blood levels of CA-125
and is retreated if an elevated CA-125 level is detected.
Example 23: Treatment of Ovarian Cancer with T Cells Sortagged with
a Bispecific Agent
[0544] Single domain antibodies specific for CA-125 and CD3 epsilon
are produced in a recombinant cell expression system. The sdAb
specific for CD3 epsilon is produced with a sortase recognition
motif incorporated at the C-terminus. Click chemistry handles are
installed at the N-termini of each sdAb using sortase. The two
sdAbs are then conjugated together to produce a bispecific
antibody. Human T cells obtained from a patient with stage III
ovarian cancer are expanded in culture with CD3-CD28 beads and then
incubated with sortase and the bispecific antibody and are
transferred back into the patient after surgery. Treatment is
repeated weekly for 12 weeks. The effect of the administered cells
on residual cancer and metastases is monitored. The patient is
monitored for blood levels of CA-125 and is retreated if an
elevated CA-125 level is detected.
Example 24: Treatment of Leukemia with Sortagged T Cells
[0545] Human T cells obtained from a patient with B-cell acute
lymphoblastic leukemia are expanded for 10 days in culture with
anti-CD3/anti-CD28 beads, then sortagged with an scFv that binds to
CD19 and transferred into the patient. The effect of the
administered cells on the leukemia is monitored.
Example 25: Treatment of Leukemia with Sortagged T Cells
[0546] Human T cells obtained from a patient with B-cell acute
lymphoblastic leukemia are genetically modified to express a
chimeric antigen cell receptor (anti-CD19 single chain Fv region
fused to the transmembrane and intracellular domain of TCR,
containing signaling domains of CD3-zeta and CD28). The cells are
expanded for 10 days in culture with anti-CD3/anti-CD28 beads, then
sortagged with an scFv that binds to CD22, and transferred into the
patient. The effect of the administered cells on the leukemia is
monitored.
Example 26: Treatment of Leukemia with Sortagged T Cells
[0547] Human T cells obtained from a patient with B-cell acute
lymphoblastic leukemia are genetically modified to express a
chimeric antigen cell receptor (anti-CD22 single chain Fv region
fused to the transmembrane and intracellular domain of TCR,
containing signaling domains of CD3-zeta and CD28). The cells are
expanded for 10 days in culture with anti-CD3/anti-CD28 beads, then
sortagged with an scFv that binds to CD19, and transferred into the
patient. The effect of the administered cells on the leukemia is
monitored.
Example 27: Clinical Trial of Treatment of Ovarian Cancer with
Anti-CA125 Antibody Conjugated T Cells
[0548] Patients with advanced ovarian cancer are randomized to
receive, following surgery, either (1) standard adjuvant
chemotherapy with platinum-taxane alone; (2) standard adjuvant
chemotherapy with platinum-taxane and, in addition, therapy with
intravenously administered autologous T cells conjugated with an
anti-CA-125 antibody using sortase without genetic modification; or
(3) standard adjuvant chemotherapy with platinum-taxane and, in
addition, therapy with both intravenously and intraperitoneally
administered autologous T cells conjugated with an anti-CA-125
antibody using sortase without genetic modification.
Progression-free survival and 5 year survival rates of patients in
the three treatment groups are monitored and compared.
Example 28: Sortase Conjugation of Red Blood Cells with Anti-PD-L1
Agent
[0549] Human red blood cells are obtained from a patient with
B-cell chronic lymphoblastic leukemia or from an immunocompatible
donor and incubated with sortase and an agent comprising (i) the
extracellular domain of PD-1 and (ii) a sortase recognition
sequence. Red blood cells are transferred into the patient after
sortagging. The effect of the administered cells on the leukemia is
monitored.
Example 29: Sortase Conjugation of Red Blood Cells with Anti-PD-1
Agent
[0550] Human red blood cells obtained from a patient with B-cell
chronic lymphoblastic leukemia or from an immunocompatible donor
are incubated with sortase and an agent comprising an scFv that
binds to PD-1 and that has been modified to comprise a sortase
recognition sequence. Red blood cells are transferred into the
patient after sortagging. The effect of the administered cells on
the leukemia is monitored.
Example 30: Treatment of Multiple Myeloma with Anti-CS1 or
Anti-BCMA Conjugated Red Blood Cells
[0551] Red blood cells are isolated from a myeloma patient or from
an immunocompatible donor. The cells are incubated with sortase and
an anti-CS-1 or anti-BCMA antibody that has been modified to
comprise a sortase recognition sequence, and are then transferred
into the patient. The treatment is repeated weekly for 12 weeks.
The effect of the administered cells on the multiple myeloma is
monitored.
Example 31: Prevention of Metastasis with TRAIL-ES Conjugated
RBCs
[0552] Red blood cells are isolated from a patient recently
diagnosed with stage III colon carcinoma or obtained from an
immunocompatible donor. The cells are incubated with sortase and an
anti-CS-1 or anti-BCMA antibody that has been modified to comprise
a sortase recognition sequence, and are then transferred into the
patient. The treatment is repeated weekly for 12 weeks following
colonic resection. The patient is monitored for presence of local
recurrence or metastasis.
Example 32: Treatment of EAE Using Red Blood Cells Sortagged with
Peptide Fragment of Myelin Basic Protein
[0553] Sortase is used to conjugate either myelin basic protein
(MBP) that has been modified to comprise a sortase recognition
sequence or ovalbumin that has been modified to comprise a sortase
recognition sequence to non-genetically modified RBCs obtained from
SJL mice.
[0554] Eight SJL mice are injected intravenously (iv) via tail vein
with 1.times.10.sup.8 RBC coupled to mouse MBP (MBP-RBC). Control
mice receive 1.times.10.sup.8 ovalbumin-coupled RBCs (OV-RBC), also
prepared using sortase. One week later, all animals are immunized
with syngeneic spinal cord homogenate in an emulsion in complete
Freund's adjuvant to induce EAE according to standard methods.
[0555] Animals are weighed and examined daily from Day 7.
Neurological deficit is graded according to the following scale:
mild, a flaccid tail for 2 or more days with associated weight
loss; or severe, definite paralysis, often with scissoring of the
hind limbs. Animals are sacrificed 27 days after initial
immunization. Brains are removed and fixed in 10% Formalin.
Sections are made, stained with hematoxylin and eosin and scored on
a scale from 1-5 in a blinded manner, according to the extent of
meningeal inflammation and lymphocyte cuffing.
[0556] The clinical severity of EAE, pathologic severity of EAE,
and weight loss are compared between the MBP-RBC and control
groups. A lower level of clinical severity, lower level of
pathological severity, and/or reduced weight loss (or weight gain
instead of weight loss) in the MBC-RBC treated group as compared
with the controls is evidence of effective inhibition of EAE.
Example 33: Treatment of Melanoma Using Sortagged T Cells
[0557] T cells isolated from a patient diagnosed with melanoma or
obtained from an immunocompatible donor are expanded and sortagged
with an antibody to a melanoma antigen, for example MART-1, and two
checkpoint inhibitors, for example ipilimumab (anti-CTLA4) and
anti-PD1 nivolumab. The sortagged T cells are administered to a
patient with melanoma. The effect of the administered cells on the
melanoma is monitored.
Example 34: Treatment of HER2 Positive Breast Cancer Using
Sortagged Red Blood Cells
[0558] RBCs isolated from a patient diagnosed with HER2 positive
breast cancer or obtained from an immunocompatible donor are
sortagged with Herceptin, anti-PD1 antibody, and anti-VEGFR2
antibody and are administered to the patient with HER2 positive
breast cancer. The effect of the administered cells on the cancer
is monitored.
Example 35: Treatment of HER2 Positive Breast Cancer Using
Sortagged Red Blood Cells
[0559] RBCs isolated from a patient diagnosed with HER2 positive
breast cancer or obtained from an immunocompatible donor are
sortagged with TRAIL and Herceptin and administered as an adjunct
to standard chemotherapy/Herceptin therapy of HER-2 positive breast
cancer. The effect of the administered cells on the cancer is
monitored.
Example 36: Treatment of Relapsed/Refractory B-Precursor ALL
[0560] Relapsed/refractory B-precursor ALL in adult patients is an
aggressive malignant disease with dismal prognosis and unmet
medical need. Red blood cells sortagged to carry blinatumomab (a
bispecific single-chain antibody construct designed to link B cells
and T cells resulting in T cell activation and a cytotoxic T cell
response against CD19 expressing cells) are administered to adult
patients with relapsed/refractory B-precursor ALL. Patients receive
up to five monthly cycles of intravenous RBC-blinatumomab
treatment.
Example 37: Sortase-Catalyzed Installation of VHHs on Toxoplasma
gondii Allows Cell-Specific Targeting
[0561] As described above, modification of CD8 T cells through
sortagging does not obviously interfere with cytotoxic functions.
Invasion of host cells by parasites represents another type of
cell-cell interaction. To investigate whether Toxoplasma gondii
tachyzoites modified using sortase would be able to invade cells,
we sortagged parasites with TAMRA-modified LPETG peptides and
incubated them together with human foreskin fibroblasts.
Tachyzoites (20 to 40 million per milliliter) were incubated with
500 .mu.M TAMRA-LPETG and 20 .mu.M Ca2+-independent sortase A (same
as used in Example 11) in HHE (Hanks buffer+Hepes+EDTA) for 15
minutes. Parasites were then washed and incubated with human
foreskin fibroblasts (HFF). Sortagged parasites were visualized by
fluorescence microscopy and their ability to invade the fibroblasts
was monitored and recorded. We found that the sortagged parasites
were perfectly capable of invading fibroblasts (FIG. 8(A); video
available upon request).
[0562] To address whether Toxoplasma gondii could be targeted to
specific cells, we sortagged parasites with biotin or biotin plus
Enhancer or VHH7 and incubated them with wild type splenocytes.
Toxoplasma gondii tachyzoites were incubated with or without 50
.mu.M enhancer-LPETG or VHH7-LPETG and 20 .mu.M Ca2+-independent
sortase A (same as used in Example 11) in HHE. After 20 minutes,
biotin-LPETG was added for 15 minutes. Parasites were then washed
and incubated with red-cell depleted splenocytes for 1 hour at a
multiplicity of infection of 5. Cells were then washed and stained
with a CD19-specific antibody and fluorescently labeled
streptavidin. The percentages of CD19+ and CD19.sup.- cells that
were biotin positive (indicative of invasion by sortagged T.
gondii) were quantified by FACS. The histogram in FIG. 8(B) shows
the percentage of cells that were positive for sortagged T. gondii
within the CD19 negative or positive populations. Sortagging of
VHH7 to Toxoplasma gondii resulted in a dramatic increase of B
cells targeted by Toxoplasma gondii, together with a significant
decrease of binding of non-B cells suggesting very selective
targeting. In addition, it enhanced the percentages of B cell lysed
upon infection (FIG. 8(C)). The B cells lysis assay was performed
as follows: Toxoplasma gondii tachyzoites were incubated with or
without 50 .mu.M enhancer- or VHH7-LPETG and 20 .mu.M sortase A at
room temperature in HHE buffer for 15 minutes. After washing T.
gondii was incubated together with 0.5 million magnetic
beads-purified splenic B cells from WT or class II MHC k.o. mice at
a multiplicity of infection of 5 in 100 .mu.l or complete RPMI in
96 flat bottom well plates. After 15 hours supernatants were
harvested and cell lysis was measured using CytoTox 96
Non-Radioactive Cytotoxicity Assay kit (Promega, cat. G1781)
according to manufacturer's instructions.
[0563] These results support the feasibility of using sortagging to
target pathogens (e.g., cytolytic pathogens) to cells of interest,
e.g., cancer cells.
[0564] The foregoing written specification is considered to be
sufficient to enable one skilled in the art to practice the
invention. Various modifications of the invention in addition to
those shown and described herein will become apparent to those
skilled in the art from the foregoing description and fall within
the scope of the appended claims. The advantages and objects of the
invention are not necessarily encompassed by each embodiment of the
invention. Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments described herein, which
fall within the scope of the claims. The scope of the present
invention is not to be limited by or to embodiments or examples
described above.
[0565] Section headings used herein are not to be construed as
limiting in any way. It is expressly contemplated that subject
matter presented under any section heading may be applicable to any
aspect or embodiment described herein.
[0566] Embodiments or aspects herein may be directed to any agent,
composition, article, kit, and/or method described herein. It is
contemplated that any one or more embodiments or aspects can be
freely combined with any one or more other embodiments or aspects
whenever appropriate. For example, any combination of two or more
agents, compositions, articles, kits, and/or methods that are not
mutually inconsistent, is provided.
[0567] Articles such as "a", "an", "the" and the like, may mean one
or more than one unless indicated to the contrary or otherwise
evident from the context.
[0568] The phrase "and/or" as used herein in the specification and
in the claims, should be understood to mean "either or both" of the
elements so conjoined. Multiple elements listed with "and/or"
should be construed in the same fashion, i.e., "one or more" of the
elements so conjoined. Other elements may optionally be present
other than the elements specifically identified by the "and/or"
clause. As used herein in the specification and in the claims, "or"
should be understood to have the same meaning as "and/or" as
defined above. For example, when used in a list of elements, "or"
or "and/or" shall be interpreted as being inclusive, i.e., the
inclusion of at least one, but optionally more than one, of list of
elements, and, optionally, additional unlisted elements. Only terms
clearly indicative to the contrary, such as "only one of" or
"exactly one of" will refer to the inclusion of exactly one element
of a number or list of elements. Thus claims that include "or"
between one or more members of a group are considered satisfied if
one, more than one, or all of the group members are present,
employed in, or otherwise relevant to a given product or process
unless indicated to the contrary. Embodiments are provided in which
exactly one member of the group is present, employed in, or
otherwise relevant to a given product or process. Embodiments are
provided in which more than one, or all of the group members are
present, employed in, or otherwise relevant to a given product or
process. Any one or more claims may be amended to explicitly
exclude any embodiment, aspect, feature, element, or
characteristic, or any combination thereof. Any one or more claims
may be amended to exclude any agent, sortase substrate, sortase,
composition, amount, dose, administration route, cell type,
species, target, cellular marker, antigen, epitope, targeting
moiety, or combination thereof. In certain embodiments cells are
not CHO cells. In certain embodiments cells are not HEK293T cells.
In certain embodiments the sortase substrate used in a sortase
reaction does not comprise an enhanced green fluorescent protein.
In certain embodiments the sortase substrate used in a sortase
reaction does not comprise an enhanced cyan fluorescent protein
(ECFP). In certain embodiments the sortase substrate used in a
sortase reaction does not comprise an AlexaFluor. In certain
embodiments cells are not pre-incubated with sortase before being
contacted with a sortase substrate in the presence of sortase. In
certain embodiments cells are not pre-incubated with a nucleophilic
acceptor sequence, e.g., an oligoglycine, e.g., triglycine, before
being contacted with a sortase substrate in the presence of
sortase. In certain embodiments if cells are pre-incubated with
sortase and/or with a nucleophilic acceptor sequence, e.g., an
oligoglycine, e.g., triglycine, before being contacted with a
sortase substrate in the presence of sortase, such pre-incubation
is for less than 15 minutes, less than 30 minutes, less than 60
minutes, or less than 120 minutes.
[0569] Embodiments in which any one or more limitations, elements,
clauses, descriptive terms, etc., of any claim (or relevant
description from elsewhere in the specification) is introduced into
another claim are provided. For example, a claim that is dependent
on another claim may be modified to include one or more elements or
limitations found in any other claim that is dependent on the same
base claim. It is expressly contemplated that any amendment to a
genus or generic claim may be applied to any species of the genus
or any species claim that incorporates or depends on the generic
claim.
[0570] Where a claim recites a composition, methods of using the
composition as disclosed herein are provided, and methods of making
the composition according to any of the methods of making disclosed
herein are provided. Where a claim recites a method, a composition
for performing the method is provided. Where elements are presented
as lists or groups, each subgroup is also disclosed. It should also
be understood that, in general, where embodiments or aspects is/are
referred to herein as comprising particular element(s), feature(s),
agent(s), substance(s), step(s), etc., (or combinations thereof),
certain embodiments or aspects may consist of, or consist
essentially of, such element(s), feature(s), agent(s),
substance(s), step(s), etc. (or combinations thereof). It should
also be understood that, unless clearly indicated to the contrary,
in any methods claimed herein that include more than one step or
act, the order of the steps or acts of the method is not
necessarily limited to the order in which the steps or acts of the
method are recited. Any method of treatment may comprise a step of
providing a subject in need of such treatment. Any method of
treatment may comprise a step of providing a subject having a
disease for which such treatment is warranted. Any method of
treatment may comprise a step of diagnosing a subject as being in
need of such treatment. Any method of treatment may comprise a step
of diagnosing a subject as having a disease for which such
treatment is warranted.
[0571] Where ranges are given herein, embodiments in which the
endpoints are included, embodiments in which both endpoints are
excluded, and embodiments in which one endpoint is included and the
other is excluded, are provided. It should be assumed that both
endpoints are included unless indicated otherwise. Unless otherwise
indicated or otherwise evident from the context and understanding
of one of ordinary skill in the art, values that are expressed as
ranges can assume any specific value or subrange within the stated
ranges in various embodiments, to the tenth of the unit of the
lower limit of the range, unless the context clearly dictates
otherwise. "About" in reference to a numerical value generally
refers to a range of values that fall within .+-.10%, in some
embodiments .+-.5%, in some embodiments .+-.1%, in some embodiments
.+-.0.5% of the value unless otherwise stated or otherwise evident
from the context. In any embodiment in which a numerical value is
prefaced by "about", an embodiment in which the exact value is
recited is provided. Where an embodiment in which a numerical value
is not prefaced by "about" is provided, an embodiment in which the
value is prefaced by "about" is also provided. Where a range is
preceded by "about", embodiments are provided in which "about"
applies to the lower limit and to the upper limit of the range or
to either the lower or the upper limit, unless the context clearly
dictates otherwise. Where a phrase such as "at least", "up to", "no
more than", or similar phrases, precedes a series of numbers, it is
to be understood that the phrase applies to each number in the list
in various embodiments (it being understood that, depending on the
context, 100% of a value, e.g., a value expressed as a percentage,
may be an upper limit), unless the context clearly dictates
otherwise. For example, "at least 1, 2, or 3" should be understood
to mean "at least 1, at least 2, or at least 3" in various
embodiments. It will also be understood that any and all reasonable
lower limits and upper limits are expressly contemplated.
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 138 <210> SEQ ID NO 1 <211> LENGTH: 206
<212> TYPE: PRT <213> ORGANISM: S. aureus <400>
SEQUENCE: 1 Met Lys Lys Trp Thr Asn Arg Leu Met Thr Ile Ala Gly Val
Val Leu 1 5 10 15 Ile Leu Val Ala Ala Tyr Leu Phe Ala Lys Pro His
Ile Asp Asn Tyr 20 25 30 Leu His Asp Lys Asp Lys Asp Glu Lys Ile
Glu Gln Tyr Asp Lys Asn 35 40 45 Val Lys Glu Gln Ala Ser Lys Asp
Asn Lys Gln Gln Ala Lys Pro Gln 50 55 60 Ile Pro Lys Asp Lys Ser
Lys Val Ala Gly Tyr Ile Glu Ile Pro Asp 65 70 75 80 Ala Asp Ile Lys
Glu Pro Val Tyr Pro Gly Pro Ala Thr Pro Glu Gln 85 90 95 Leu Asn
Arg Gly Val Ser Phe Ala Glu Glu Asn Glu Ser Leu Asp Asp 100 105 110
Gln Asn Ile Ser Ile Ala Gly His Thr Phe Ile Asp Arg Pro Asn Tyr 115
120 125 Gln Phe Thr Asn Leu Lys Ala Ala Lys Lys Gly Ser Met Val Tyr
Phe 130 135 140 Lys Val Gly Asn Glu Thr Arg Lys Tyr Lys Met Thr Ser
Ile Arg Asp 145 150 155 160 Val Lys Pro Thr Asp Val Glu Val Leu Asp
Glu Gln Lys Gly Lys Asp 165 170 175 Lys Gln Leu Thr Leu Ile Thr Cys
Asp Asp Tyr Asn Glu Lys Thr Gly 180 185 190 Val Trp Glu Lys Arg Lys
Ile Phe Val Ala Thr Glu Val Lys 195 200 205 <210> SEQ ID NO 2
<211> LENGTH: 206 <212> TYPE: PRT <213> ORGANISM:
S. aureus <400> SEQUENCE: 2 Met Lys Lys Trp Thr Asn Arg Leu
Met Thr Ile Ala Gly Val Val Leu 1 5 10 15 Ile Leu Val Ala Ala Tyr
Leu Phe Ala Lys Pro His Ile Asp Asn Tyr 20 25 30 Leu His Asp Lys
Asp Lys Asp Glu Lys Ile Glu Gln Tyr Asp Lys Asn 35 40 45 Val Lys
Glu Gln Ala Ser Lys Asp Lys Lys Gln Gln Ala Lys Pro Gln 50 55 60
Ile Pro Lys Asp Lys Ser Lys Val Ala Gly Tyr Ile Glu Ile Pro Asp 65
70 75 80 Ala Asp Ile Lys Glu Pro Val Tyr Pro Gly Pro Ala Thr Pro
Glu Gln 85 90 95 Leu Asn Arg Gly Val Ser Phe Ala Glu Glu Asn Glu
Ser Leu Asp Asp 100 105 110 Gln Asn Ile Ser Ile Ala Gly His Thr Phe
Ile Asp Arg Pro Asn Tyr 115 120 125 Gln Phe Thr Asn Leu Lys Ala Ala
Lys Lys Gly Ser Met Val Tyr Phe 130 135 140 Lys Val Gly Asn Glu Thr
Arg Lys Tyr Lys Met Thr Ser Ile Arg Asp 145 150 155 160 Val Lys Pro
Thr Asp Val Gly Val Leu Asp Glu Gln Lys Gly Lys Asp 165 170 175 Lys
Gln Leu Thr Leu Ile Thr Cys Asp Asp Tyr Asn Glu Lys Thr Gly 180 185
190 Val Trp Glu Lys Arg Lys Ile Phe Val Ala Thr Glu Val Lys 195 200
205 <210> SEQ ID NO 3 <211> LENGTH: 148 <212>
TYPE: PRT <213> ORGANISM: S. aureus <400> SEQUENCE: 3
Met Gln Ala Lys Pro Gln Ile Pro Lys Asp Lys Ser Lys Val Ala Gly 1 5
10 15 Tyr Ile Glu Ile Pro Asp Ala Asp Ile Lys Glu Pro Val Tyr Pro
Gly 20 25 30 Pro Ala Thr Arg Glu Gln Leu Asn Arg Gly Val Ser Phe
Ala Glu Glu 35 40 45 Asn Glu Ser Leu Asp Asp Gln Asn Ile Ser Ile
Ala Gly His Thr Phe 50 55 60 Ile Asp Arg Pro Asn Tyr Gln Phe Thr
Asn Leu Lys Ala Ala Lys Lys 65 70 75 80 Gly Ser Met Val Tyr Phe Lys
Val Gly Asn Glu Thr Arg Lys Tyr Lys 85 90 95 Met Thr Ser Ile Arg
Asn Val Lys Pro Thr Ala Val Glu Val Leu Asp 100 105 110 Glu Gln Lys
Gly Lys Asp Lys Gln Leu Thr Leu Ile Thr Cys Asp Asp 115 120 125 Tyr
Asn Glu Glu Thr Gly Val Trp Glu Thr Arg Lys Ile Phe Val Ala 130 135
140 Thr Glu Val Lys 145 <210> SEQ ID NO 4 <211> LENGTH:
148 <212> TYPE: PRT <213> ORGANISM: S. aureus
<400> SEQUENCE: 4 Met Gln Ala Lys Pro Gln Ile Pro Lys Asp Lys
Ser Lys Val Ala Gly 1 5 10 15 Tyr Ile Glu Ile Pro Asp Ala Asp Ile
Lys Glu Pro Val Tyr Pro Gly 20 25 30 Pro Ala Thr Arg Glu Gln Leu
Asn Arg Gly Val Ser Phe Ala Lys Glu 35 40 45 Asn Gln Ser Leu Asp
Asp Gln Asn Ile Ser Ile Ala Gly His Thr Phe 50 55 60 Ile Asp Arg
Pro Asn Tyr Gln Phe Thr Asn Leu Lys Ala Ala Lys Lys 65 70 75 80 Gly
Ser Met Val Tyr Phe Lys Val Gly Asn Glu Thr Arg Lys Tyr Lys 85 90
95 Met Thr Ser Ile Arg Asn Val Lys Pro Thr Ala Val Glu Val Leu Asp
100 105 110 Glu Gln Lys Gly Lys Asp Lys Gln Leu Thr Leu Ile Thr Cys
Asp Asp 115 120 125 Tyr Asn Glu Glu Thr Gly Val Trp Glu Thr Arg Lys
Ile Phe Val Ala 130 135 140 Thr Glu Val Lys 145 <210> SEQ ID
NO 5 <211> LENGTH: 7 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Polypeptide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (2)..(2)
<223> OTHER INFORMATION: Xaa is Val or Ile <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(4)..(4) <223> OTHER INFORMATION: Xaa can be any naturally
occurring amino acid <400> SEQUENCE: 5 Asp Xaa Glu Xaa Asn
Pro Gly 1 5 <210> SEQ ID NO 6 <211> LENGTH: 22
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <400> SEQUENCE: 6 Val Lys Gln Thr Leu Asn Phe Asp
Leu Leu Lys Leu Ala Gly Asp Val 1 5 10 15 Glu Ser Asn Pro Gly Pro
20 <210> SEQ ID NO 7 <211> LENGTH: 156 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide
<400> SEQUENCE: 7 Met Gln Ala Lys Pro Gln Ile Pro Lys Asp Lys
Ser Lys Val Ala Gly 1 5 10 15 Tyr Ile Glu Ile Pro Asp Ala Asp Ile
Lys Glu Pro Val Tyr Pro Gly 20 25 30 Pro Ala Thr Arg Glu Gln Leu
Asn Arg Gly Val Ser Phe Ala Lys Glu 35 40 45 Asn Gln Ser Leu Asp
Asp Gln Asn Ile Ser Ile Ala Gly His Thr Phe 50 55 60 Ile Asp Arg
Pro Asn Tyr Gln Phe Thr Asn Leu Lys Ala Ala Lys Lys 65 70 75 80 Gly
Ser Met Val Tyr Phe Lys Val Gly Asn Glu Thr Arg Lys Tyr Lys 85 90
95 Met Thr Ser Ile Arg Asn Val Lys Pro Thr Ala Val Glu Val Leu Asp
100 105 110 Glu Gln Lys Gly Lys Asp Lys Gln Leu Thr Leu Ile Thr Cys
Asp Asp 115 120 125 Tyr Asn Glu Glu Thr Gly Val Trp Glu Thr Arg Lys
Ile Phe Val Ala 130 135 140 Thr Glu Val Lys Leu Glu His His His His
His His 145 150 155 <210> SEQ ID NO 8 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (3)..(3) <223> OTHER INFORMATION: Xaa
can be any naturally occurring amino acid <400> SEQUENCE: 8
Leu Pro Xaa Thr Gly 1 5 <210> SEQ ID NO 9 <211> LENGTH:
6 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (3)..(3) <223> OTHER INFORMATION: Xaa
can be any naturally occurring amino acid <400> SEQUENCE: 9
Leu Pro Xaa Thr Gly Gly 1 5 <210> SEQ ID NO 10 <211>
LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Polypeptide <400> SEQUENCE: 10 Leu Pro Glu Thr Gly
Gly 1 5 <210> SEQ ID NO 11 <211> LENGTH: 6 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (4)..(4) <223> OTHER INFORMATION: Xaa can be any
naturally occurring amino acid <400> SEQUENCE: 11 Lys Leu Pro
Xaa Thr Gly 1 5 <210> SEQ ID NO 12 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <400> SEQUENCE: 12 Leu Pro Glu Thr Gly 1 5
<210> SEQ ID NO 13 <211> LENGTH: 8 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 13 Ser Ile Ile Asn Phe Glu Lys Leu 1 5 <210> SEQ ID
NO 14 <211> LENGTH: 5 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 14
Leu Pro Glu Thr Gly 1 5 <210> SEQ ID NO 15 <211>
LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Polypeptide <400> SEQUENCE: 15 Leu Pro Glu Thr Gly
1 5 <210> SEQ ID NO 16 <211> LENGTH: 4 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (4)..(4) <223> OTHER INFORMATION: Ser can be
replaced by Thr, Gly or Ala <400> SEQUENCE: 16 Gly Gly Gly
Ser 1 <210> SEQ ID NO 17 <211> LENGTH: 5 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide
<400> SEQUENCE: 17 Gly Gly Gly Gly Ser 1 5 <210> SEQ ID
NO 18 <211> LENGTH: 5 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Polypeptide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (2)..(2)
<223> OTHER INFORMATION: Xaa can be any naturally occurring
amino acid <400> SEQUENCE: 18 Leu Xaa Pro Thr Gly 1 5
<210> SEQ ID NO 19 <211> LENGTH: 6 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 19 His His His His His His 1 5 <210> SEQ ID NO 20
<400> SEQUENCE: 20 000 <210> SEQ ID NO 21 <400>
SEQUENCE: 21 000 <210> SEQ ID NO 22 <211> LENGTH: 4
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <400> SEQUENCE: 22 Gly Gly Gly Gly 1 <210>
SEQ ID NO 23 <211> LENGTH: 5 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 23 Gly Gly Gly Gly Gly 1 5 <210> SEQ ID NO 24
<211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 24 Ala Ala
Ala Ala 1 <210> SEQ ID NO 25 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <400> SEQUENCE: 25 Ala Ala Ala Ala Ala 1 5
<210> SEQ ID NO 26 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 26 Leu Pro Lys Thr Gly 1 5 <210> SEQ ID NO 27
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 27 Leu Pro
Ala Thr Gly 1 5 <210> SEQ ID NO 28 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <400> SEQUENCE: 28 Leu Pro Asn Thr Gly 1 5
<210> SEQ ID NO 29 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(3)..(3) <223> OTHER INFORMATION: Xaa can be any naturally
occurring amino acid <400> SEQUENCE: 29 Leu Pro Xaa Ala Gly 1
5 <210> SEQ ID NO 30 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 30 Leu Pro Asn Ala Gly 1 5 <210> SEQ ID NO 31
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Polypeptide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (3)..(3) <223>
OTHER INFORMATION: Xaa can be any naturally occurring amino acid
<400> SEQUENCE: 31 Leu Pro Xaa Thr Ala 1 5 <210> SEQ ID
NO 32 <211> LENGTH: 5 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 32
Leu Pro Asn Thr Ala 1 5 <210> SEQ ID NO 33 <211>
LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Polypeptide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (3)..(3) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid
<400> SEQUENCE: 33 Leu Gly Xaa Thr Gly 1 5 <210> SEQ ID
NO 34 <211> LENGTH: 5 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 34
Leu Gly Ala Thr Gly 1 5 <210> SEQ ID NO 35 <211>
LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Polypeptide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (3)..(3) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid
<400> SEQUENCE: 35 Ile Pro Xaa Thr Gly 1 5 <210> SEQ ID
NO 36 <211> LENGTH: 5 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 36
Ile Pro Asn Thr Gly 1 5 <210> SEQ ID NO 37 <211>
LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Polypeptide <400> SEQUENCE: 37 Ile Pro Glu Thr Gly
1 5 <210> SEQ ID NO 38 <211> LENGTH: 5 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (3)..(3) <223> OTHER INFORMATION: Xaa can be any
naturally occurring amino acid <400> SEQUENCE: 38 Thr Leu Xaa
Thr Cys 1 5 <210> SEQ ID NO 39 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (3)..(3) <223> OTHER INFORMATION: Xaa
is Gln or Lys <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(4) <223> OTHER
INFORMATION: Xaa is Thr or Ser <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (5)..(5) <223>
OTHER INFORMATION: Xaa is Asn, Gly or Ser <400> SEQUENCE: 39
Asn Pro Xaa Xaa Xaa 1 5 <210> SEQ ID NO 40 <211>
LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Polypeptide <400> SEQUENCE: 40 Asn Pro Gln Thr Asn
1 5 <210> SEQ ID NO 41 <211> LENGTH: 5 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide
<400> SEQUENCE: 41 Asn Pro Lys Thr Gly 1 5 <210> SEQ ID
NO 42 <211> LENGTH: 5 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 42
Asn Ser Lys Thr Ala 1 5 <210> SEQ ID NO 43 <211>
LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Polypeptide <400> SEQUENCE: 43 Asn Pro Gln Thr Gly
1 5 <210> SEQ ID NO 44 <211> LENGTH: 5 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide
<400> SEQUENCE: 44 Asn Ala Lys Thr Asn 1 5 <210> SEQ ID
NO 45 <211> LENGTH: 5 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 45
Asn Pro Gln Ser Ser 1 5 <210> SEQ ID NO 46 <211>
LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Polypeptide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (3)..(3) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid
<400> SEQUENCE: 46 Asn Ala Xaa Thr Gly 1 5 <210> SEQ ID
NO 47 <211> LENGTH: 5 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Polypeptide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (3)..(3)
<223> OTHER INFORMATION: Xaa can be any naturally occurring
amino acid <400> SEQUENCE: 47 Leu Ala Xaa Thr Gly 1 5
<210> SEQ ID NO 48 <211> LENGTH: 6 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 48 Gln Val Pro Thr Gly Val 1 5 <210> SEQ ID NO 49
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Polypeptide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (3)..(3) <223>
OTHER INFORMATION: Xaa can be any naturally occurring amino acid
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (5)..(5) <223> OTHER INFORMATION: Xaa is Ala or Ser
<400> SEQUENCE: 49 Leu Pro Xaa Thr Xaa 1 5 <210> SEQ ID
NO 50 <211> LENGTH: 5 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 50
Leu Pro Ser Thr Ser 1 5 <210> SEQ ID NO 51 <211>
LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Polypeptide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (3)..(3) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (5)..(5) <223> OTHER INFORMATION: Xaa can be any
naturally occurring amino acid <400> SEQUENCE: 51 Ser Pro Xaa
Thr Xaa 1 5 <210> SEQ ID NO 52 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (3)..(3) <223> OTHER INFORMATION: Xaa
can be any naturally occurring amino acid <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (5)..(5)
<223> OTHER INFORMATION: Xaa can be any naturally occurring
amino acid <400> SEQUENCE: 52 Leu Ser Xaa Thr Xaa 1 5
<210> SEQ ID NO 53 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(3)..(3) <223> OTHER INFORMATION: Xaa is Asp, Glu, Ala, Asn,
Gln, Lys or Arg <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (5)..(5) <223> OTHER
INFORMATION: Xaa is Gly or Ser <400> SEQUENCE: 53 Asn Pro Xaa
Thr Xaa 1 5 <210> SEQ ID NO 54 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (3)..(3) <223> OTHER INFORMATION: Xaa
is Asp, Glu, Ala, Asn, Gln, Lys or Arg <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (5)..(5)
<223> OTHER INFORMATION: Xaa is Gly or Ser <400>
SEQUENCE: 54 Val Pro Xaa Thr Xaa 1 5 <210> SEQ ID NO 55
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Polypeptide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (3)..(3) <223>
OTHER INFORMATION: Xaa is Asp, Glu, Ala, Asn, Gln, Lys or Arg
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (5)..(5) <223> OTHER INFORMATION: Xaa is Gly or Ser
<400> SEQUENCE: 55 Ile Pro Xaa Thr Xaa 1 5 <210> SEQ ID
NO 56 <211> LENGTH: 5 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Polypeptide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (3)..(3)
<223> OTHER INFORMATION: Xaa is Asp, Glu, Ala, Asn, Gln, Lys
or Arg <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (5)..(5) <223> OTHER INFORMATION: Xaa
is Gly or Ser <400> SEQUENCE: 56 Tyr Pro Xaa Arg Xaa 1 5
<210> SEQ ID NO 57 <211> LENGTH: 4 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 57 Leu Pro Lys Thr 1 <210> SEQ ID NO 58 <211>
LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Polypeptide <400> SEQUENCE: 58 Leu Pro Ile Thr 1
<210> SEQ ID NO 59 <211> LENGTH: 4 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 59 Leu Pro Asp Thr 1 <210> SEQ ID NO 60 <211>
LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Polypeptide <400> SEQUENCE: 60 Ser Pro Lys Thr 1
<210> SEQ ID NO 61 <211> LENGTH: 4 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 61 Leu Ala Glu Thr 1 <210> SEQ ID NO 62 <211>
LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Polypeptide <400> SEQUENCE: 62 Leu Ala Ala Thr 1
<210> SEQ ID NO 63 <211> LENGTH: 4 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 63 Leu Ala Ser Thr 1 <210> SEQ ID NO 64 <211>
LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Polypeptide <400> SEQUENCE: 64 Leu Pro Leu Thr 1
<210> SEQ ID NO 65 <211> LENGTH: 4 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 65 Leu Ser Arg Thr 1 <210> SEQ ID NO 66 <211>
LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Polypeptide <400> SEQUENCE: 66 Leu Pro Glu Thr 1
<210> SEQ ID NO 67 <211> LENGTH: 4 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 67 Val Pro Asp Thr 1 <210> SEQ ID NO 68 <211>
LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Polypeptide <400> SEQUENCE: 68 Ile Pro Gln Thr 1
<210> SEQ ID NO 69 <211> LENGTH: 4 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 69 Tyr Pro Arg Arg 1 <210> SEQ ID NO 70 <211>
LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Polypeptide <400> SEQUENCE: 70 Leu Pro Met Thr 1
<210> SEQ ID NO 71 <211> LENGTH: 4 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 71 Leu Ala Phe Thr 1 <210> SEQ ID NO 72 <211>
LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Polypeptide <400> SEQUENCE: 72 Leu Pro Gln Thr 1
<210> SEQ ID NO 73 <211> LENGTH: 4 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 73 Asn Ser Lys Thr 1 <210> SEQ ID NO 74 <211>
LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Polypeptide <400> SEQUENCE: 74 Asn Pro Gln Thr 1
<210> SEQ ID NO 75 <211> LENGTH: 4 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 75 Asn Ala Lys Thr 1 <210> SEQ ID NO 76 <211>
LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Polypeptide <400> SEQUENCE: 76 Asn Pro Gln Ser 1
<210> SEQ ID NO 77 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 77 Leu Pro Lys Thr Gly 1 5 <210> SEQ ID NO 78
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 78 Leu Pro
Ile Thr Gly 1 5 <210> SEQ ID NO 79 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <400> SEQUENCE: 79 Leu Pro Asp Thr Ala 1 5
<210> SEQ ID NO 80 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 80 Ser Pro Lys Thr Gly 1 5 <210> SEQ ID NO 81
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 81 Leu Ala
Glu Thr Gly 1 5 <210> SEQ ID NO 82 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <400> SEQUENCE: 82 Leu Ala Ala Thr Gly 1 5
<210> SEQ ID NO 83 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 83 Leu Ala His Thr Gly 1 5 <210> SEQ ID NO 84
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 84 Leu Ala
Ser Thr Gly 1 5 <210> SEQ ID NO 85 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <400> SEQUENCE: 85 Leu Ala Glu Thr Gly 1 5
<210> SEQ ID NO 86 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 86 Leu Pro Leu Thr Gly 1 5 <210> SEQ ID NO 87
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 87 Leu Ser
Arg Thr Gly 1 5 <210> SEQ ID NO 88 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <400> SEQUENCE: 88 Leu Pro Glu Thr Gly 1 5
<210> SEQ ID NO 89 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 89 Val Pro Asp Thr Gly 1 5 <210> SEQ ID NO 90
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 90 Ile Pro
Gln Thr Gly 1 5 <210> SEQ ID NO 91 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <400> SEQUENCE: 91 Tyr Pro Arg Arg Gly 1 5
<210> SEQ ID NO 92 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 92 Leu Pro Met Thr Gly 1 5 <210> SEQ ID NO 93
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 93 Leu Pro
Leu Thr Gly 1 5 <210> SEQ ID NO 94 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <400> SEQUENCE: 94 Leu Ala Phe Thr Gly 1 5
<210> SEQ ID NO 95 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 95 Leu Pro Gln Thr Ser 1 5 <210> SEQ ID NO 96
<211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 96 Leu Pro
Ser Thr 1 <210> SEQ ID NO 97 <211> LENGTH: 4
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <400> SEQUENCE: 97 Arg Ala Lys Arg 1 <210>
SEQ ID NO 98 <211> LENGTH: 9 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 98 Thr Thr Cys Cys Gly Leu Arg Gln Tyr 1 5 <210>
SEQ ID NO 99 <211> LENGTH: 303 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 99 Ile Lys Gly Gly Leu Phe Ala Asp Ile Ala Ser His Pro
Trp Gln Ala 1 5 10 15 Ala Ile Phe Ala Lys His His Arg Arg Gly Gly
Glu Arg Phe Leu Cys 20 25 30 Gly Gly Ile Leu Ile Ser Ser Cys Trp
Ile Leu Ser Ala Ala His Cys 35 40 45 Phe Gln Gln Gln Gln Gln Glu
Glu Glu Glu Glu Arg Arg Arg Arg Arg 50 55 60 Phe Phe Phe Phe Phe
Pro Pro Pro Pro Pro Pro His His Leu Thr Val 65 70 75 80 Ile Leu Gly
Arg Thr Tyr Arg Val Val Pro Gly Glu Glu Glu Gln Lys 85 90 95 Phe
Glu Val Glu Lys Tyr Ile Val His Lys Glu Phe Asp Asp Asp Thr 100 105
110 Tyr Asp Asn Asp Ile Ala Leu Leu Gln Leu Lys Ser Ser Ser Ser Ser
115 120 125 Asp Asp Asp Asp Asp Ser Ser Ser Ser Ser Ser Ser Ser Ser
Ser Arg 130 135 140 Arg Arg Arg Arg Cys Ala Gln Glu Ser Ser Val Val
Arg Thr Val Cys 145 150 155 160 Leu Pro Pro Ala Asp Leu Gln Leu Pro
Asp Trp Thr Glu Cys Glu Leu 165 170 175 Ser Gly Tyr Gly Lys His Glu
Ala Leu Ser Pro Phe Tyr Ser Glu Arg 180 185 190 Leu Lys Glu Ala His
Val Arg Leu Tyr Pro Ser Ser Arg Cys Thr Thr 195 200 205 Thr Ser Ser
Ser Gln Gln Gln His Leu Leu Asn Arg Thr Val Thr Asp 210 215 220 Asn
Met Leu Cys Ala Gly Asp Thr Thr Thr Arg Arg Arg Ser Ser Ser 225 230
235 240 Asn Asn Asn Leu His Asp Ala Cys Gln Gly Asp Ser Gly Gly Pro
Leu 245 250 255 Val Cys Leu Asn Asp Gly Arg Met Thr Leu Val Gly Ile
Ile Ser Trp 260 265 270 Gly Leu Gly Cys Gly Gly Gln Gln Lys Asp Val
Pro Gly Val Tyr Thr 275 280 285 Lys Val Thr Asn Tyr Leu Asp Trp Ile
Arg Asp Asn Met Arg Pro 290 295 300 <210> SEQ ID NO 100
<211> LENGTH: 255 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 100 Val
Val Gly Gly Glu Asp Ala Lys Pro Gly Gln Phe Pro Trp Gln Val 1 5 10
15 Val Leu Asn Gly Lys Val Asp Ala Phe Cys Gly Gly Ser Ile Val Asn
20 25 30 Glu Lys Trp Ile Val Thr Ala Ala His Cys Val Glu Glu Thr
Thr Gly 35 40 45 Val Lys Ile Thr Val Val Ala Gly Glu His Asn Ile
Glu Glu Thr Glu 50 55 60 His Thr Glu Gln Lys Arg Asn Val Ile Arg
Ile Ile Pro His His Asn 65 70 75 80 Tyr Asn Asn Asn Ala Ala Ala Ala
Ala Ala Ile Asn Lys Tyr Asn His 85 90 95 Asp Ile Ala Leu Leu Glu
Leu Asp Glu Pro Leu Val Leu Asn Ser Tyr 100 105 110 Val Thr Pro Ile
Cys Ile Ala Asp Lys Glu Tyr Thr Thr Thr Asn Asn 115 120 125 Asn Ile
Ile Ile Phe Leu Lys Phe Gly Ser Gly Tyr Val Ser Gly Trp 130 135 140
Gly Arg Val Phe His Lys Gly Arg Ser Ala Leu Val Leu Gln Tyr Leu 145
150 155 160 Arg Val Pro Leu Val Asp Arg Ala Thr Cys Leu Arg Ser Thr
Lys Phe 165 170 175 Thr Ile Tyr Asn Asn Met Phe Cys Ala Gly Gly Phe
Phe His Glu Gly 180 185 190 Gly Gly Arg Arg Asp Ser Cys Gln Gly Asp
Ser Gly Gly Pro His Val 195 200 205 Thr Glu Val Glu Gly Thr Ser Phe
Leu Thr Gly Ile Ile Ser Trp Gly 210 215 220 Glu Glu Cys Ala Ala Met
Met Lys Gly Lys Tyr Gly Ile Tyr Thr Lys 225 230 235 240 Val Ser Arg
Tyr Val Asn Trp Ile Lys Glu Lys Thr Lys Leu Thr 245 250 255
<210> SEQ ID NO 101 <211> LENGTH: 57 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 101 Met Thr Cys Asn Ile Lys Asn Gly Arg Cys Glu Gln Phe
Cys Lys Asn 1 5 10 15 Ser Ala Asp Asn Lys Val Val Cys Ser Cys Thr
Glu Gly Tyr Arg Leu 20 25 30 Ala Glu Asn Gln Lys Ser Cys Glu Pro
Ala Val Pro Phe Pro Cys Gly 35 40 45 Arg Val Ser Val Ser Gln Thr
Ser Lys 50 55 <210> SEQ ID NO 102 <211> LENGTH: 496
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <400> SEQUENCE: 102 Glu Phe Ala Arg Pro Cys Ile
Pro Lys Ser Phe Gly Tyr Ser Ser Val 1 5 10 15 Val Cys Val Cys Asn
Ala Thr Tyr Cys Asp Ser Phe Asp Pro Pro Ala 20 25 30 Leu Gly Thr
Phe Ser Arg Tyr Glu Ser Thr Arg Ser Gly Arg Arg Met 35 40 45 Glu
Leu Ser Met Gly Pro Ile Gln Ala Asn His Thr Gly Thr Gly Leu 50 55
60 Leu Leu Thr Leu Gln Pro Glu Gln Lys Phe Gln Lys Val Lys Gly Phe
65 70 75 80 Gly Gly Ala Met Thr Asp Ala Ala Ala Leu Asn Ile Leu Ala
Leu Ser 85 90 95 Pro Pro Ala Gln Asn Leu Leu Leu Lys Ser Tyr Phe
Ser Glu Glu Gly 100 105 110 Ile Gly Tyr Asn Ile Ile Arg Val Pro Met
Ala Ser Cys Asp Phe Ser 115 120 125 Ile Arg Thr Tyr Thr Tyr Ala Asp
Thr Pro Asp Asp Phe Gln Leu His 130 135 140 Asn Phe Ser Leu Pro Glu
Glu Asp Thr Lys Leu Lys Ile Pro Leu Ile 145 150 155 160 His Arg Ala
Leu Gln Leu Ala Gln Arg Pro Val Ser Leu Leu Ala Ser 165 170 175 Pro
Trp Thr Ser Pro Thr Trp Leu Lys Thr Asn Gly Ala Val Asn Gly 180 185
190 Lys Gly Ser Leu Lys Gly Gln Pro Gly Asp Ile Tyr His Gln Thr Trp
195 200 205 Ala Arg Tyr Phe Val Lys Phe Leu Asp Ala Tyr Ala Glu His
Lys Leu 210 215 220 Gln Phe Trp Ala Val Thr Ala Glu Asn Glu Pro Ser
Ala Gly Leu Leu 225 230 235 240 Ser Gly Tyr Pro Phe Gln Cys Leu Gly
Phe Thr Pro Glu His Gln Arg 245 250 255 Asp Phe Ile Ala Arg Asp Leu
Gly Pro Thr Leu Ala Asn Ser Thr His 260 265 270 His Asn Val Arg Leu
Leu Met Leu Asp Asp Gln Arg Leu Leu Leu Pro 275 280 285 His Trp Ala
Lys Val Val Leu Thr Asp Pro Glu Ala Ala Lys Tyr Val 290 295 300 His
Gly Ile Ala Val His Trp Tyr Leu Asp Phe Leu Ala Pro Ala Lys 305 310
315 320 Ala Thr Leu Gly Glu Thr His Arg Leu Phe Pro Asn Thr Met Leu
Phe 325 330 335 Ala Ser Glu Ala Cys Val Gly Ser Lys Phe Trp Glu Gln
Ser Val Arg 340 345 350 Leu Gly Ser Trp Asp Arg Gly Met Gln Tyr Ser
His Ser Ile Ile Thr 355 360 365 Asn Leu Leu Tyr His Val Val Gly Trp
Thr Asp Trp Asn Leu Ala Leu 370 375 380 Asn Pro Glu Gly Gly Pro Asn
Trp Val Arg Asn Phe Val Asp Ser Pro 385 390 395 400 Ile Ile Val Asp
Ile Thr Lys Asp Thr Phe Tyr Lys Gln Pro Met Phe 405 410 415 Tyr His
Leu Gly His Phe Ser Lys Phe Ile Pro Glu Gly Ser Gln Arg 420 425 430
Val Gly Leu Val Ala Ser Gln Lys Asn Asp Leu Asp Ala Val Ala Leu 435
440 445 Met His Pro Asp Gly Ser Ala Val Val Val Val Leu Asn Arg Ser
Ser 450 455 460 Lys Asp Val Pro Leu Thr Ile Lys Asp Pro Ala Val Gly
Phe Leu Glu 465 470 475 480 Thr Ile Ser Pro Gly Tyr Ser Ile His Thr
Tyr Leu Trp His Arg Gln 485 490 495 <210> SEQ ID NO 103
<211> LENGTH: 390 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 103 Leu
Asp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly Trp Leu His Trp 1 5 10
15 Glu Arg Phe Met Cys Asn Leu Asp Cys Gln Glu Glu Pro Asp Ser Cys
20 25 30 Ile Ser Glu Lys Leu Phe Met Glu Met Ala Glu Leu Met Val
Ser Glu 35 40 45 Gly Trp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys Ile
Asp Asp Cys Trp 50 55 60 Met Ala Pro Gln Arg Asp Ser Glu Gly Arg
Leu Gln Ala Asp Pro Gln 65 70 75 80 Arg Phe Pro His Gly Ile Arg Gln
Leu Ala Asn Tyr Val His Ser Lys 85 90 95 Gly Leu Lys Leu Gly Ile
Tyr Ala Asp Val Gly Asn Lys Thr Cys Ala 100 105 110 Gly Phe Pro Gly
Ser Phe Gly Tyr Tyr Asp Ile Asp Ala Gln Thr Phe 115 120 125 Ala Asp
Trp Gly Val Asp Leu Leu Lys Phe Asp Gly Cys Tyr Cys Asp 130 135 140
Ser Leu Glu Asn Leu Ala Asp Gly Tyr Lys His Met Ser Leu Ala Leu 145
150 155 160 Asn Arg Thr Gly Arg Ser Ile Val Tyr Ser Cys Glu Trp Pro
Leu Tyr 165 170 175 Met Trp Pro Phe Gln Lys Pro Asn Tyr Thr Glu Ile
Arg Gln Tyr Cys 180 185 190 Asn His Trp Arg Asn Phe Ala Asp Ile Asp
Asp Ser Trp Lys Ser Ile 195 200 205 Lys Ser Ile Leu Asp Trp Thr Ser
Phe Asn Gln Glu Arg Ile Val Asp 210 215 220 Val Ala Gly Pro Gly Gly
Trp Asn Asp Pro Asp Met Leu Val Ile Gly 225 230 235 240 Asn Phe Gly
Leu Ser Trp Asn Gln Gln Val Thr Gln Met Ala Leu Trp 245 250 255 Ala
Ile Met Ala Ala Pro Leu Phe Met Ser Asn Asp Leu Arg His Ile 260 265
270 Ser Pro Gln Ala Lys Ala Leu Leu Gln Asp Lys Asp Val Ile Ala Ile
275 280 285 Asn Gln Asp Pro Leu Gly Lys Gln Gly Tyr Gln Leu Arg Gln
Gly Asp 290 295 300 Asn Phe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu
Ala Trp Ala Val 305 310 315 320 Ala Met Ile Asn Arg Gln Glu Ile Gly
Gly Pro Arg Ser Tyr Thr Ile 325 330 335 Ala Val Ala Ser Leu Gly Lys
Gly Val Ala Cys Asn Pro Ala Cys Phe 340 345 350 Ile Thr Gln Leu Leu
Pro Val Lys Arg Lys Leu Gly Phe Tyr Glu Trp 355 360 365 Thr Ser Arg
Leu Arg Ser His Ile Asn Pro Thr Gly Thr Val Leu Leu 370 375 380 Gln
Leu Glu Asn Thr Met 385 390 <210> SEQ ID NO 104 <211>
LENGTH: 479 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Polypeptide <400> SEQUENCE: 104 Arg Pro Pro Asn Ile
Val Leu Ile Phe Ala Asp Asp Leu Gly Tyr Gly 1 5 10 15 Asp Leu Gly
Cys Tyr Gly His Pro Ser Ser Thr Thr Pro Asn Leu Asp 20 25 30 Gln
Leu Ala Ala Gly Gly Leu Arg Phe Thr Asp Phe Tyr Val Pro Val 35 40
45 Ser Leu Pro Ser Arg Ala Ala Leu Leu Thr Gly Arg Leu Pro Val Arg
50 55 60 Met Gly Met Tyr Pro Gly Val Leu Val Pro Ser Ser Arg Gly
Gly Leu 65 70 75 80 Pro Leu Glu Glu Val Thr Val Ala Glu Val Leu Ala
Ala Arg Gly Tyr 85 90 95 Leu Thr Gly Met Ala Gly Lys Trp His Leu
Gly Val Gly Pro Glu Gly 100 105 110 Ala Phe Leu Pro Pro His Gln Gly
Phe His Arg Phe Leu Gly Ile Pro 115 120 125 Tyr Ser His Asp Gln Gly
Pro Cys Gln Asn Leu Thr Cys Phe Pro Pro 130 135 140 Ala Thr Pro Cys
Asp Gly Gly Cys Asp Gln Gly Leu Val Pro Ile Pro 145 150 155 160 Leu
Leu Ala Asn Leu Ser Val Glu Ala Gln Pro Pro Trp Leu Pro Gly 165 170
175 Leu Glu Ala Arg Tyr Met Ala Phe Ala His Asp Leu Met Ala Asp Ala
180 185 190 Gln Arg Gln Asp Arg Pro Phe Phe Leu Tyr Tyr Ala Ser His
His Thr 195 200 205 His Tyr Pro Gln Phe Ser Gly Gln Ser Phe Ala Glu
Arg Ser Gly Arg 210 215 220 Gly Pro Phe Gly Asp Ser Leu Met Glu Leu
Asp Ala Ala Val Gly Thr 225 230 235 240 Leu Met Thr Ala Ile Gly Asp
Leu Gly Leu Leu Glu Glu Thr Leu Val 245 250 255 Ile Phe Thr Ala Asp
Asn Gly Pro Glu Thr Met Arg Met Ser Arg Gly 260 265 270 Gly Cys Ser
Gly Leu Leu Arg Cys Gly Lys Gly Thr Thr Tyr Glu Gly 275 280 285 Gly
Val Arg Glu Pro Ala Leu Ala Phe Trp Pro Gly His Ile Ala Pro 290 295
300 Gly Val Thr His Glu Leu Ala Ser Ser Leu Asp Leu Leu Pro Thr Leu
305 310 315 320 Ala Ala Leu Ala Gly Ala Pro Leu Pro Asn Val Thr Leu
Asp Gly Phe 325 330 335 Asp Leu Ser Pro Leu Leu Leu Gly Thr Gly Lys
Ser Pro Arg Gln Ser 340 345 350 Leu Phe Phe Tyr Pro Ser Tyr Pro Asp
Glu Val Arg Gly Val Phe Ala 355 360 365 Val Arg Thr Gly Lys Tyr Lys
Ala His Phe Phe Thr Gln Gly Ser Ala 370 375 380 His Ser Asp Thr Thr
Ala Asp Pro Ala Cys His Ala Ser Ser Ser Leu 385 390 395 400 Thr Ala
His Glu Pro Pro Leu Leu Tyr Asp Leu Ser Lys Asp Pro Gly 405 410 415
Glu Asn Tyr Asn Leu Leu Gly Ala Thr Pro Glu Val Leu Gln Ala Leu 420
425 430 Lys Gln Leu Gln Leu Leu Lys Ala Gln Leu Asp Ala Ala Val Thr
Phe 435 440 445 Gly Pro Ser Gln Val Ala Arg Gly Glu Asp Pro Ala Leu
Gln Ile Cys 450 455 460 Cys His Pro Gly Cys Thr Pro Arg Pro Ala Cys
Cys His Cys Pro 465 470 475 <210> SEQ ID NO 105 <211>
LENGTH: 474 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Polypeptide <400> SEQUENCE: 105 Ser Arg Pro Pro His
Leu Val Phe Leu Leu Ala Asp Asp Leu Gly Trp 1 5 10 15 Asn Asp Val
Gly Phe His Gly Ser Arg Ile Arg Thr Pro His Leu Asp 20 25 30 Ala
Leu Ala Ala Gly Gly Val Leu Leu Asp Asn Tyr Tyr Thr Gln Pro 35 40
45 Leu Thr Pro Ser Arg Ser Gln Leu Leu Thr Gly Arg Tyr Gln Ile Arg
50 55 60 Thr Gly Leu Gln His Gln Ile Ile Trp Pro Cys Gln Pro Ser
Cys Val 65 70 75 80 Pro Leu Asp Glu Lys Leu Leu Pro Gln Leu Leu Lys
Glu Ala Gly Tyr 85 90 95 Thr Thr His Met Val Gly Lys Trp His Leu
Gly Met Tyr Arg Lys Glu 100 105 110 Cys Leu Pro Thr Arg Arg Gly Phe
Asp Thr Tyr Phe Gly Tyr Leu Leu 115 120 125 Gly Ser Glu Asp Tyr Tyr
Ser His Glu Arg Cys Thr Leu Ile Asp Ala 130 135 140 Leu Asn Val Thr
Arg Cys Ala Leu Asp Phe Arg Asp Gly Glu Glu Val 145 150 155 160 Ala
Thr Gly Tyr Lys Asn Met Tyr Ser Thr Asn Ile Phe Thr Lys Arg 165 170
175 Ala Ile Ala Leu Ile Thr Asn His Pro Pro Glu Lys Pro Leu Phe Leu
180 185 190 Tyr Leu Ala Leu Gln Ser Val His Glu Pro Leu Gln Val Pro
Glu Glu 195 200 205 Tyr Leu Lys Pro Tyr Asp Phe Ile Gln Asp Lys Asn
Arg His His Tyr 210 215 220 Ala Gly Met Val Ser Leu Met Asp Glu Ala
Val Gly Asn Val Thr Ala 225 230 235 240 Ala Leu Lys Ser Ser Gly Leu
Trp Asn Asn Thr Val Phe Ile Phe Ser 245 250 255 Thr Asp Asn Gly Gly
Gln Thr Leu Ala Gly Gly Asn Asn Trp Pro Leu 260 265 270 Arg Gly Arg
Lys Trp Ser Leu Trp Glu Gly Gly Val Arg Gly Val Gly 275 280 285 Phe
Val Ala Ser Pro Leu Leu Lys Gln Lys Gly Val Lys Asn Arg Glu 290 295
300 Leu Ile His Ile Ser Asp Trp Leu Pro Thr Leu Val Lys Leu Ala Arg
305 310 315 320 Gly His Thr Asn Gly Thr Lys Pro Leu Asp Gly Phe Asp
Val Trp Lys 325 330 335 Thr Ile Ser Glu Gly Ser Pro Ser Pro Arg Ile
Glu Leu Leu His Asn 340 345 350 Ile Asp Pro Asn Phe Val Asp Ser Ser
Pro Cys Ser Ala Phe Asn Thr 355 360 365 Ser Val His Ala Ala Ile Arg
His Gly Asn Trp Lys Leu Leu Thr Gly 370 375 380 Tyr Pro Gly Cys Gly
Tyr Trp Phe Pro Pro Pro Ser Gln Tyr Asn Val 385 390 395 400 Ser Glu
Ile Pro Ser Ser Asp Pro Pro Thr Lys Thr Leu Trp Leu Phe 405 410 415
Asp Ile Asp Arg Asp Pro Glu Glu Arg His Asp Leu Ser Arg Glu Tyr 420
425 430 Pro His Ile Val Thr Lys Leu Leu Ser Arg Leu Gln Phe Tyr His
Lys 435 440 445 His Ser Val Pro Val Tyr Phe Pro Ala Gln Asp Pro Arg
Cys Asp Pro 450 455 460 Lys Ala Thr Gly Val Trp Gly Pro Trp Met 465
470 <210> SEQ ID NO 106 <211> LENGTH: 492 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide
<400> SEQUENCE: 106 Leu Trp Pro Trp Pro Gln Asn Phe Gln Thr
Ser Asp Gln Arg Tyr Val 1 5 10 15 Leu Tyr Pro Asn Asn Phe Gln Phe
Gln Tyr Asp Val Ser Ser Ala Ala 20 25 30 Gln Pro Gly Cys Ser Val
Leu Asp Glu Ala Phe Gln Arg Tyr Arg Asp 35 40 45 Leu Leu Phe Gly
Thr Leu Glu Lys Asn Val Leu Val Val Ser Val Val 50 55 60 Thr Pro
Gly Cys Asn Gln Leu Pro Thr Leu Glu Ser Val Glu Asn Tyr 65 70 75 80
Thr Leu Thr Ile Asn Asp Asp Gln Cys Leu Leu Leu Ser Glu Thr Val 85
90 95 Trp Gly Ala Leu Arg Gly Leu Glu Thr Phe Ser Gln Leu Val Trp
Lys 100 105 110 Ser Ala Glu Gly Thr Phe Phe Ile Asn Lys Thr Glu Ile
Glu Asp Phe 115 120 125 Pro Arg Phe Pro His Arg Gly Leu Leu Leu Asp
Thr Ser Arg His Tyr 130 135 140 Leu Pro Leu Ser Ser Ile Leu Asp Thr
Leu Asp Val Met Ala Tyr Asn 145 150 155 160 Lys Leu Asn Val Phe His
Trp His Leu Val Asp Asp Pro Ser Phe Pro 165 170 175 Tyr Glu Ser Phe
Thr Phe Pro Glu Leu Met Arg Lys Gly Ser Tyr Asn 180 185 190 Pro Val
Thr His Ile Tyr Thr Ala Gln Asp Val Lys Glu Val Ile Glu 195 200 205
Tyr Ala Arg Leu Arg Gly Ile Arg Val Leu Ala Glu Phe Asp Thr Pro 210
215 220 Gly His Thr Leu Ser Trp Gly Pro Gly Ile Pro Gly Leu Leu Thr
Pro 225 230 235 240 Cys Tyr Ser Gly Ser Glu Pro Ser Gly Thr Phe Gly
Pro Val Asn Pro 245 250 255 Ser Leu Asn Asn Thr Tyr Glu Phe Met Ser
Thr Phe Phe Leu Glu Val 260 265 270 Ser Ser Val Phe Pro Asp Phe Tyr
Leu His Leu Gly Gly Asp Glu Val 275 280 285 Asp Phe Thr Cys Trp Lys
Ser Asn Pro Glu Ile Gln Asp Phe Met Arg 290 295 300 Lys Lys Gly Phe
Gly Glu Asp Phe Lys Gln Leu Glu Ser Phe Tyr Ile 305 310 315 320 Gln
Thr Leu Leu Asp Ile Val Ser Ser Tyr Gly Lys Gly Tyr Val Val 325 330
335 Trp Gln Glu Val Phe Asp Asn Lys Val Lys Ile Gln Pro Asp Thr Ile
340 345 350 Ile Gln Val Trp Arg Glu Asp Ile Pro Val Asn Tyr Met Lys
Glu Leu 355 360 365 Glu Leu Val Thr Lys Ala Gly Phe Arg Ala Leu Leu
Ser Ala Pro Trp 370 375 380 Tyr Leu Asn Arg Ile Ser Tyr Gly Pro Asp
Trp Lys Asp Phe Tyr Val 385 390 395 400 Val Glu Pro Leu Ala Phe Glu
Gly Thr Pro Glu Gln Lys Ala Leu Val 405 410 415 Ile Gly Gly Glu Ala
Cys Met Trp Gly Glu Tyr Val Asp Asn Thr Asn 420 425 430 Leu Val Pro
Arg Leu Trp Pro Arg Ala Gly Ala Val Ala Glu Arg Leu 435 440 445 Trp
Ser Asn Lys Leu Thr Ser Asp Leu Thr Phe Ala Tyr Glu Arg Leu 450 455
460 Ser His Phe Arg Cys Glu Leu Leu Arg Arg Gly Val Gln Ala Gln Pro
465 470 475 480 Leu Asn Val Gly Phe Cys Glu Gln Glu Phe Glu Gln 485
490 <210> SEQ ID NO 107 <211> LENGTH: 492 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide
<400> SEQUENCE: 107 Leu Trp Pro Trp Pro Gln Asn Phe Gln Thr
Ser Asp Gln Arg Tyr Val 1 5 10 15 Leu Tyr Pro Asn Asn Phe Gln Phe
Gln Tyr Asp Val Ser Ser Ala Ala 20 25 30 Gln Pro Gly Cys Ser Val
Leu Asp Glu Ala Phe Gln Arg Tyr Arg Asp 35 40 45 Leu Leu Phe Gly
Thr Leu Glu Lys Asn Val Leu Val Val Ser Val Val 50 55 60 Thr Pro
Gly Cys Asn Gln Leu Pro Thr Leu Glu Ser Val Glu Asn Tyr 65 70 75 80
Thr Leu Thr Ile Asn Asp Asp Gln Cys Leu Leu Leu Ser Glu Thr Val 85
90 95 Trp Gly Ala Leu Arg Gly Leu Glu Thr Phe Ser Gln Leu Val Trp
Lys 100 105 110 Ser Ala Glu Gly Thr Phe Phe Ile Asn Lys Thr Glu Ile
Glu Asp Phe 115 120 125 Pro Arg Phe Pro His Arg Gly Leu Leu Leu Asp
Thr Ser Arg His Tyr 130 135 140 Leu Pro Leu Ser Ser Ile Leu Asp Thr
Leu Asp Val Met Ala Tyr Asn 145 150 155 160 Lys Leu Asn Val Phe His
Trp His Leu Val Asp Asp Pro Ser Phe Pro 165 170 175 Tyr Glu Ser Phe
Thr Phe Pro Glu Leu Met Arg Lys Gly Ser Tyr Asn 180 185 190 Pro Val
Thr His Ile Tyr Thr Ala Gln Asp Val Lys Glu Val Ile Glu 195 200 205
Tyr Ala Arg Leu Arg Gly Ile Arg Val Leu Ala Glu Phe Asp Thr Pro 210
215 220 Gly His Thr Leu Ser Trp Gly Pro Gly Ile Pro Gly Leu Leu Thr
Pro 225 230 235 240 Cys Tyr Ser Gly Ser Glu Pro Ser Gly Thr Phe Gly
Pro Val Asn Pro 245 250 255 Ser Leu Asn Asn Thr Tyr Glu Phe Met Ser
Thr Phe Phe Leu Glu Val 260 265 270 Ser Ser Val Phe Pro Asp Phe Tyr
Leu His Leu Gly Gly Asp Glu Val 275 280 285 Asp Phe Thr Cys Trp Lys
Ser Asn Pro Glu Ile Gln Asp Phe Met Arg 290 295 300 Lys Lys Gly Phe
Gly Glu Asp Phe Lys Gln Leu Glu Ser Phe Tyr Ile 305 310 315 320 Gln
Thr Leu Leu Asp Ile Val Ser Ser Tyr Gly Lys Gly Tyr Val Val 325 330
335 Trp Gln Glu Val Phe Asp Asn Lys Val Lys Ile Gln Pro Asp Thr Ile
340 345 350 Ile Gln Val Trp Arg Glu Asp Ile Pro Val Asn Tyr Met Lys
Glu Leu 355 360 365 Glu Leu Val Thr Lys Ala Gly Phe Arg Ala Leu Leu
Ser Ala Pro Trp 370 375 380 Tyr Leu Asn Arg Ile Ser Tyr Gly Pro Asp
Trp Lys Asp Phe Tyr Val 385 390 395 400 Val Glu Pro Leu Ala Phe Glu
Gly Thr Pro Glu Gln Lys Ala Leu Val 405 410 415 Ile Gly Gly Glu Ala
Cys Met Trp Gly Glu Tyr Val Asp Asn Thr Asn 420 425 430 Leu Val Pro
Arg Leu Trp Pro Arg Ala Gly Ala Val Ala Glu Arg Leu 435 440 445 Trp
Ser Asn Lys Leu Thr Ser Asp Leu Thr Phe Ala Tyr Glu Arg Leu 450 455
460 Ser His Phe Arg Cys Glu Leu Leu Arg Arg Gly Val Gln Ala Gln Pro
465 470 475 480 Leu Asn Val Gly Phe Cys Glu Gln Glu Phe Glu Gln 485
490 <210> SEQ ID NO 108 <211> LENGTH: 480 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide
<400> SEQUENCE: 108 Pro Ala Leu Trp Pro Leu Pro Leu Ser Val
Lys Met Thr Pro Asn Leu 1 5 10 15 Leu His Leu Ala Pro Glu Asn Phe
Tyr Ile Ser His Ser Pro Asn Ser 20 25 30 Thr Ala Gly Pro Ser Cys
Thr Leu Leu Glu Glu Ala Phe Arg Arg Tyr 35 40 45 His Gly Tyr Ile
Phe Gly Thr Gln Val Gln Gln Leu Leu Val Ser Ile 50 55 60 Thr Leu
Gln Ser Glu Cys Asp Ala Phe Pro Asn Ile Ser Ser Asp Glu 65 70 75 80
Ser Tyr Thr Leu Leu Val Lys Glu Pro Val Ala Val Leu Lys Ala Asn 85
90 95 Arg Val Trp Gly Ala Leu Arg Gly Leu Glu Thr Phe Ser Gln Leu
Val 100 105 110 Tyr Gln Asp Ser Tyr Gly Thr Phe Thr Ile Asn Glu Ser
Thr Ile Ile 115 120 125 Asp Ser Pro Arg Phe Ser His Arg Gly Ile Leu
Ile Asp Thr Ser Arg 130 135 140 His Tyr Leu Pro Val Lys Ile Ile Leu
Lys Thr Leu Asp Ala Met Ala 145 150 155 160 Phe Asn Lys Phe Asn Val
Leu His Trp His Ile Val Asp Asp Gln Ser 165 170 175 Phe Pro Tyr Gln
Ser Ile Thr Phe Pro Glu Leu Ser Asn Lys Gly Ser 180 185 190 Tyr Ser
Leu Ser His Val Tyr Thr Pro Asn Asp Val Arg Met Val Ile 195 200 205
Glu Tyr Ala Arg Leu Arg Gly Ile Arg Val Leu Pro Glu Phe Asp Thr 210
215 220 Pro Gly His Thr Leu Ser Trp Gly Lys Gly Gln Lys Asp Leu Leu
Thr 225 230 235 240 Pro Cys Tyr Ser Asp Ser Phe Gly Pro Ile Asn Pro
Thr Leu Asn Thr 245 250 255 Thr Tyr Ser Phe Leu Thr Thr Phe Phe Lys
Glu Ile Ser Glu Val Phe 260 265 270 Pro Asp Gln Phe Ile His Leu Gly
Gly Asp Glu Val Glu Phe Lys Cys 275 280 285 Trp Glu Ser Asn Pro Lys
Ile Gln Asp Phe Met Arg Gln Lys Gly Phe 290 295 300 Gly Thr Asp Phe
Lys Lys Leu Glu Ser Phe Tyr Ile Gln Lys Val Leu 305 310 315 320 Asp
Ile Ile Ala Thr Ile Asn Lys Gly Ser Ile Val Trp Gln Glu Val 325 330
335 Phe Asp Asp Lys Ala Lys Leu Ala Pro Gly Thr Ile Val Glu Val Trp
340 345 350 Lys Asp Ser Ala Tyr Pro Glu Glu Leu Ser Arg Val Thr Ala
Ser Gly 355 360 365 Phe Pro Val Ile Leu Ser Ala Pro Trp Tyr Leu Asp
Leu Ile Ser Tyr 370 375 380 Gly Gln Asp Trp Arg Lys Tyr Tyr Lys Val
Glu Pro Leu Asp Phe Gly 385 390 395 400 Gly Thr Gln Lys Gln Lys Gln
Leu Phe Ile Gly Gly Glu Ala Cys Leu 405 410 415 Trp Gly Glu Tyr Val
Asp Ala Thr Asn Leu Thr Pro Arg Leu Trp Pro 420 425 430 Arg Ala Ser
Ala Val Gly Glu Arg Leu Trp Ser Ser Lys Asp Val Arg 435 440 445 Asp
Met Asp Asp Ala Tyr Asp Arg Leu Thr Arg His Arg Cys Arg Met 450 455
460 Val Glu Arg Gly Ile Ala Ala Gln Pro Leu Tyr Ala Gly Tyr Cys Asn
465 470 475 480 <210> SEQ ID NO 109 <211> LENGTH: 481
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <400> SEQUENCE: 109 Pro Ala Leu Trp Pro Leu Pro
Leu Ser Val Lys Met Thr Pro Asn Leu 1 5 10 15 Leu His Leu Ala Pro
Glu Asn Phe Tyr Ile Ser His Ser Pro Asn Ser 20 25 30 Thr Ala Gly
Pro Ser Cys Thr Leu Leu Glu Glu Ala Phe Arg Arg Tyr 35 40 45 His
Gly Tyr Ile Phe Gly Thr Gln Val Gln Gln Leu Leu Val Ser Ile 50 55
60 Thr Leu Gln Ser Glu Cys Asp Ala Phe Pro Asn Ile Ser Ser Asp Glu
65 70 75 80 Ser Tyr Thr Leu Leu Val Lys Glu Pro Val Ala Val Leu Lys
Ala Asn 85 90 95 Arg Val Trp Gly Ala Leu Arg Gly Leu Glu Thr Phe
Ser Gln Leu Val 100 105 110 Tyr Gln Asp Ser Tyr Gly Thr Phe Thr Ile
Asn Glu Ser Thr Ile Ile 115 120 125 Asp Ser Pro Arg Phe Ser His Arg
Gly Ile Leu Ile Asp Thr Ser Arg 130 135 140 His Tyr Leu Pro Val Lys
Ile Ile Leu Lys Thr Leu Asp Ala Met Ala 145 150 155 160 Phe Asn Lys
Phe Asn Val Leu His Trp His Ile Val Asp Asp Gln Ser 165 170 175 Phe
Pro Tyr Gln Ser Ile Thr Phe Pro Glu Leu Ser Asn Lys Gly Ser 180 185
190 Tyr Ser Leu Ser His Val Tyr Thr Pro Asn Asp Val Arg Met Val Ile
195 200 205 Glu Tyr Ala Arg Leu Arg Gly Ile Arg Val Leu Pro Glu Phe
Asp Thr 210 215 220 Pro Gly His Thr Leu Ser Trp Gly Lys Gly Gln Lys
Asp Leu Leu Thr 225 230 235 240 Pro Cys Tyr Ser Leu Asp Ser Phe Gly
Pro Ile Asn Pro Thr Leu Asn 245 250 255 Thr Thr Tyr Ser Phe Leu Thr
Thr Phe Phe Lys Glu Ile Ser Glu Val 260 265 270 Phe Pro Asp Gln Phe
Ile His Leu Gly Gly Asp Glu Val Glu Phe Lys 275 280 285 Cys Trp Glu
Ser Asn Pro Lys Ile Gln Asp Phe Met Arg Gln Lys Gly 290 295 300 Phe
Gly Thr Asp Phe Lys Lys Leu Glu Ser Phe Tyr Ile Gln Lys Val 305 310
315 320 Leu Asp Ile Ile Ala Thr Ile Asn Lys Gly Ser Ile Val Trp Gln
Glu 325 330 335 Val Phe Asp Asp Lys Ala Lys Leu Ala Pro Gly Thr Ile
Val Glu Val 340 345 350 Trp Lys Asp Ser Ala Tyr Pro Glu Glu Leu Ser
Arg Val Thr Ala Ser 355 360 365 Gly Phe Pro Val Ile Leu Ser Ala Pro
Trp Tyr Leu Asp Leu Ile Ser 370 375 380 Tyr Gly Gln Asp Trp Arg Lys
Tyr Tyr Lys Val Glu Pro Leu Asp Phe 385 390 395 400 Gly Gly Thr Gln
Lys Gln Lys Gln Leu Phe Ile Gly Gly Glu Ala Cys 405 410 415 Leu Trp
Gly Glu Tyr Val Asp Ala Thr Asn Leu Thr Pro Arg Leu Trp 420 425 430
Pro Arg Ala Ser Ala Val Gly Glu Arg Leu Trp Ser Ser Lys Asp Val 435
440 445 Arg Asp Met Asp Asp Ala Tyr Asp Arg Leu Thr Arg His Arg Cys
Arg 450 455 460 Met Val Glu Arg Gly Ile Ala Ala Gln Pro Leu Tyr Ala
Gly Tyr Cys 465 470 475 480 Asn <210> SEQ ID NO 110
<211> LENGTH: 492 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 110 Leu
Trp Pro Trp Pro Gln Asn Phe Gln Thr Ser Asp Gln Arg Tyr Val 1 5 10
15 Leu Tyr Pro Asn Asn Phe Gln Phe Gln Tyr Asp Val Ser Ser Ala Ala
20 25 30 Gln Pro Gly Cys Ser Val Leu Asp Glu Ala Phe Gln Arg Tyr
Arg Asp 35 40 45 Leu Leu Phe Gly Thr Leu Glu Lys Asn Val Leu Val
Val Ser Val Val 50 55 60 Thr Pro Gly Cys Asn Gln Leu Pro Thr Leu
Glu Ser Val Glu Asn Tyr 65 70 75 80 Thr Leu Thr Ile Asn Asp Asp Gln
Cys Leu Leu Leu Ser Glu Thr Val 85 90 95 Trp Gly Ala Leu Arg Gly
Leu Glu Thr Phe Ser Gln Leu Val Trp Lys 100 105 110 Ser Ala Glu Gly
Thr Phe Phe Ile Asn Lys Thr Glu Ile Glu Asp Phe 115 120 125 Pro Arg
Phe Pro His Arg Gly Leu Leu Leu Asp Thr Ser Arg His Tyr 130 135 140
Leu Pro Leu Ser Ser Ile Leu Asp Thr Leu Asp Val Met Ala Tyr Asn 145
150 155 160 Lys Leu Asn Val Phe His Trp His Leu Val Asp Asp Pro Ser
Phe Pro 165 170 175 Tyr Glu Ser Phe Thr Phe Pro Glu Leu Met Arg Lys
Gly Ser Tyr Asn 180 185 190 Pro Val Thr His Ile Tyr Thr Ala Gln Asp
Val Lys Glu Val Ile Glu 195 200 205 Tyr Ala Arg Leu Arg Gly Ile Arg
Val Leu Ala Glu Phe Asp Thr Pro 210 215 220 Gly His Thr Leu Ser Trp
Gly Pro Gly Ile Pro Gly Leu Leu Thr Pro 225 230 235 240 Cys Tyr Ser
Gly Ser Glu Pro Ser Gly Thr Phe Gly Pro Val Asn Pro 245 250 255 Ser
Leu Asn Asn Thr Tyr Glu Phe Met Ser Thr Phe Phe Leu Glu Val 260 265
270 Ser Ser Val Phe Pro Asp Phe Tyr Leu His Leu Gly Gly Asp Glu Val
275 280 285 Asp Phe Thr Cys Trp Lys Ser Asn Pro Glu Ile Gln Asp Phe
Met Arg 290 295 300 Lys Lys Gly Phe Gly Glu Asp Phe Lys Gln Leu Glu
Ser Phe Tyr Ile 305 310 315 320 Gln Thr Leu Leu Asp Ile Val Ser Ser
Tyr Gly Lys Gly Tyr Val Val 325 330 335 Trp Gln Glu Val Phe Asp Asn
Lys Val Lys Ile Gln Pro Asp Thr Ile 340 345 350 Ile Gln Val Trp Arg
Glu Asp Ile Pro Val Asn Tyr Met Lys Glu Leu 355 360 365 Glu Leu Val
Thr Lys Ala Gly Phe Arg Ala Leu Leu Ser Ala Pro Trp 370 375 380 Tyr
Leu Asn Arg Ile Ser Tyr Gly Pro Asp Trp Lys Asp Phe Tyr Val 385 390
395 400 Val Glu Pro Leu Ala Phe Glu Gly Thr Pro Glu Gln Lys Ala Leu
Val 405 410 415 Ile Gly Gly Glu Ala Cys Met Trp Gly Glu Tyr Val Asp
Asn Thr Asn 420 425 430 Leu Val Pro Arg Leu Trp Pro Arg Ala Gly Ala
Val Ala Glu Arg Leu 435 440 445 Trp Ser Asn Lys Leu Thr Ser Asp Leu
Thr Phe Ala Tyr Glu Arg Leu 450 455 460 Ser His Phe Arg Cys Glu Leu
Leu Arg Arg Gly Val Gln Ala Gln Pro 465 470 475 480 Leu Asn Val Gly
Phe Cys Glu Gln Glu Phe Glu Gln 485 490 <210> SEQ ID NO 111
<211> LENGTH: 307 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 111 Val
Pro Trp Phe Pro Arg Thr Ile Gln Glu Leu Asp Arg Phe Ala Asn 1 5 10
15 Gln Ile Leu Ser Tyr Gly Ala Glu Leu Asp Ala Asp His Pro Gly Phe
20 25 30 Lys Asp Pro Val Tyr Arg Ala Arg Arg Lys Gln Phe Ala Asp
Ile Ala 35 40 45 Tyr Asn Tyr Arg His Gly Gln Pro Ile Pro Arg Val
Glu Tyr Met Glu 50 55 60 Glu Glu Lys Lys Thr Trp Gly Thr Val Phe
Lys Thr Leu Lys Ser Leu 65 70 75 80 Tyr Lys Thr His Ala Cys Tyr Glu
Tyr Asn His Ile Phe Pro Leu Leu 85 90 95 Glu Lys Tyr Cys Gly Phe
His Glu Asp Asn Ile Pro Gln Leu Glu Asp 100 105 110 Val Ser Gln Phe
Leu Gln Thr Cys Thr Gly Phe Arg Leu Arg Pro Val 115 120 125 Ala Gly
Leu Leu Ser Ser Arg Asp Phe Leu Gly Gly Leu Ala Phe Arg 130 135 140
Val Phe His Cys Thr Gln Tyr Ile Arg His Gly Ser Lys Pro Met Tyr 145
150 155 160 Thr Pro Glu Pro Asp Ile Cys His Glu Leu Leu Gly His Val
Pro Leu 165 170 175 Phe Ser Asp Arg Ser Phe Ala Gln Phe Ser Gln Glu
Ile Gly Leu Ala 180 185 190 Ser Leu Gly Ala Pro Asp Glu Tyr Ile Glu
Lys Leu Ala Thr Ile Tyr 195 200 205 Trp Phe Thr Val Glu Phe Gly Leu
Cys Lys Gln Gly Asp Ser Ile Lys 210 215 220 Ala Tyr Gly Ala Gly Leu
Leu Ser Ser Phe Gly Glu Leu Gln Tyr Cys 225 230 235 240 Leu Ser Glu
Lys Pro Lys Leu Leu Pro Leu Glu Leu Glu Lys Thr Ala 245 250 255 Ile
Gln Asn Tyr Thr Val Thr Glu Phe Gln Pro Leu Tyr Tyr Val Ala 260 265
270 Glu Ser Phe Asn Asp Ala Lys Glu Lys Val Arg Asn Phe Ala Ala Thr
275 280 285 Ile Pro Arg Pro Phe Ser Val Arg Tyr Asp Pro Tyr Thr Gln
Arg Ile 290 295 300 Glu Val Leu 305 <210> SEQ ID NO 112
<211> LENGTH: 452 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 112 Ala
Pro Asp Gln Asp Glu Ile Gln Arg Leu Pro Gly Leu Ala Lys Gln 1 5 10
15 Pro Ser Phe Arg Gln Tyr Ser Gly Tyr Leu Lys Ser Ser Gly Ser Lys
20 25 30 His Leu His Tyr Trp Phe Val Glu Ser Gln Lys Asp Pro Glu
Asn Ser 35 40 45 Pro Val Val Leu Trp Leu Asn Gly Gly Pro Gly Cys
Ser Ser Leu Asp 50 55 60 Gly Leu Leu Thr Glu His Gly Pro Phe Leu
Val Gln Pro Asp Gly Val 65 70 75 80 Thr Leu Glu Tyr Asn Pro Tyr Ser
Trp Asn Leu Ile Ala Asn Val Leu 85 90 95 Tyr Leu Glu Ser Pro Ala
Gly Val Gly Phe Ser Tyr Ser Asp Asp Lys 100 105 110 Phe Tyr Ala Thr
Asn Asp Thr Glu Val Ala Gln Ser Asn Phe Glu Ala 115 120 125 Leu Gln
Asp Phe Phe Arg Leu Phe Pro Glu Tyr Lys Asn Asn Lys Leu 130 135 140
Phe Leu Thr Gly Glu Ser Tyr Ala Gly Ile Tyr Ile Pro Thr Leu Ala 145
150 155 160 Val Leu Val Met Gln Asp Pro Ser Met Asn Leu Gln Gly Leu
Ala Val 165 170 175 Gly Asn Gly Leu Ser Ser Tyr Glu Gln Asn Asp Asn
Ser Leu Val Tyr 180 185 190 Phe Ala Tyr Tyr His Gly Leu Leu Gly Asn
Arg Leu Trp Ser Ser Leu 195 200 205 Gln Thr His Cys Cys Ser Gln Asn
Lys Cys Asn Phe Tyr Asp Asn Lys 210 215 220 Asp Leu Glu Cys Val Thr
Asn Leu Gln Glu Val Ala Arg Ile Val Gly 225 230 235 240 Asn Ser Gly
Leu Asn Ile Tyr Asn Leu Tyr Ala Pro Cys Ala Gly Gly 245 250 255 Val
Pro Ser His Phe Arg Tyr Glu Lys Asp Thr Val Val Val Gln Asp 260 265
270 Leu Gly Asn Ile Phe Thr Arg Leu Pro Leu Lys Arg Met Trp His Gln
275 280 285 Ala Leu Leu Arg Ser Gly Asp Lys Val Arg Met Asp Pro Pro
Cys Thr 290 295 300 Asn Thr Thr Ala Ala Ser Thr Tyr Leu Asn Asn Pro
Tyr Val Arg Lys 305 310 315 320 Ala Leu Asn Ile Pro Glu Gln Leu Pro
Gln Trp Asp Met Cys Asn Phe 325 330 335 Leu Val Asn Leu Gln Tyr Arg
Arg Leu Tyr Arg Ser Met Asn Ser Gln 340 345 350 Tyr Leu Lys Leu Leu
Ser Ser Gln Lys Tyr Gln Ile Leu Leu Tyr Asn 355 360 365 Gly Asp Val
Asp Met Ala Cys Asn Phe Met Gly Asp Glu Trp Phe Val 370 375 380 Asp
Ser Leu Asn Gln Lys Met Glu Val Gln Arg Arg Pro Trp Leu Val 385 390
395 400 Lys Tyr Gly Asp Ser Gly Glu Gln Ile Ala Gly Phe Val Lys Glu
Phe 405 410 415 Ser His Ile Ala Phe Leu Thr Ile Lys Gly Ala Gly His
Met Val Pro 420 425 430 Thr Asp Lys Pro Leu Ala Ala Phe Thr Met Phe
Ser Arg Phe Leu Asn 435 440 445 Lys Gln Pro Tyr 450 <210> SEQ
ID NO 113 <211> LENGTH: 145 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 113
Leu Pro Gln Ser Phe Leu Leu Lys Cys Leu Glu Gln Val Arg Lys Ile 1 5
10 15 Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr
Lys 20 25 30 Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser
Leu Gly Ile 35 40 45 Pro Trp Ala Pro Leu Leu Ala Gly Cys Leu Ser
Gln Leu His Ser Gly 50 55 60 Leu Phe Leu Tyr Gln Gly Leu Leu Gln
Ala Leu Glu Gly Ile Ser Pro 65 70 75 80 Glu Leu Gly Pro Thr Leu Asp
Thr Leu Gln Leu Asp Val Ala Asp Phe 85 90 95 Ala Thr Thr Ile Trp
Gln Gln Met Glu Glu Leu Gly Met Met Pro Ala 100 105 110 Phe Ala Ser
Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser 115 120 125 His
Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu 130 135
140 Ala 145 <210> SEQ ID NO 114 <211> LENGTH: 105
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <400> SEQUENCE: 114 Glu His Val Asn Ala Ile Gln
Glu Ala Arg Arg Leu Leu Asn Leu Ser 1 5 10 15 Arg Asp Thr Ala Ala
Glu Met Asn Glu Thr Val Glu Val Ile Ser Glu 20 25 30 Met Phe Asp
Leu Gln Glu Pro Thr Cys Leu Gln Thr Arg Leu Glu Leu 35 40 45 Tyr
Lys Gln Gly Leu Arg Gly Ser Leu Thr Lys Leu Lys Gly Pro Leu 50 55
60 Thr Met Met Ala Ser His Tyr Lys Gln His Cys Pro Pro Thr Pro Glu
65 70 75 80 Thr Ser Cys Ala Thr Gln Ile Ile Thr Phe Glu Ser Phe Lys
Glu Asn 85 90 95 Leu Lys Asp Phe Leu Leu Val Ile Pro 100 105
<210> SEQ ID NO 115 <211> LENGTH: 165 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 115 Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg
Thr Leu Met 1 5 10 15 Leu Leu Ala Gln Met Arg Lys Ile Ser Leu Phe
Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Gly Phe Pro Gln Glu
Glu Phe Gly Asn Gln Phe Gln 35 40 45 Lys Ala Glu Thr Ile Pro Val
Leu His Glu Met Ile Gln Gln Ile Phe 50 55 60 Asn Leu Phe Ser Thr
Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu 65 70 75 80 Leu Asp Lys
Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu 85 90 95 Ala
Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys 100 105
110 Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu
115 120 125 Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val
Val Arg 130 135 140 Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn
Leu Gln Glu Ser 145 150 155 160 Leu Arg Ser Lys Glu 165 <210>
SEQ ID NO 116 <211> LENGTH: 166 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 116 Met Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser Ser
Asn Phe Gln 1 5 10 15 Cys Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg
Leu Glu Tyr Cys Leu 20 25 30 Lys Asp Arg Met Asn Phe Asp Ile Pro
Glu Glu Ile Lys Gln Leu Gln 35 40 45 Gln Phe Gln Lys Glu Asp Ala
Ala Leu Thr Ile Tyr Glu Met Leu Gln 50 55 60 Asn Ile Phe Ala Ile
Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp Asn 65 70 75 80 Glu Thr Ile
Val Glu Asn Leu Leu Ala Asn Val Tyr His Gln Ile Asn 85 90 95 His
Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe Thr 100 105
110 Arg Gly Lys Leu Met Ser Ser Leu His Leu Lys Arg Tyr Tyr Gly Arg
115 120 125 Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala
Trp Thr 130 135 140 Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe
Ile Asn Arg Leu 145 150 155 160 Thr Gly Tyr Leu Arg Asn 165
<210> SEQ ID NO 117 <211> LENGTH: 242 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 117 Met Gln Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys
Lys Tyr Phe 1 5 10 15 Asn Ala Gly His Ser Asp Val Ala Asp Asn Gly
Thr Leu Phe Leu Gly 20 25 30 Ile Leu Lys Asn Trp Lys Glu Glu Ser
Asp Arg Lys Ile Met Gln Ser 35 40 45 Gln Ile Val Ser Phe Tyr Phe
Lys Leu Phe Lys Asn Phe Lys Asp Asp 50 55 60 Gln Ser Ile Gln Lys
Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val 65 70 75 80 Lys Phe Phe
Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu 85 90 95 Thr
Asn Tyr Ser Val Thr Asp Leu Asn Val Gln Arg Lys Ala Ile Asp 100 105
110 Glu Leu Ile Gln Val Met Ala Glu Leu Gly Ala Asn Val Ser Gly Glu
115 120 125 Phe Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr Phe Asn Asp
Asn Gly 130 135 140 Thr Leu Phe Leu Gly Ile Leu Lys Asn Trp Lys Glu
Glu Ser Asp Arg 145 150 155 160 Lys Ile Met Gln Ser Gln Ile Val Ser
Phe Tyr Phe Lys Leu Phe Lys 165 170 175 Asn Phe Lys Asp Asp Gln Ser
Ile Gln Lys Ser Val Glu Thr Ile Lys 180 185 190 Glu Asp Met Asn Val
Lys Phe Phe Asn Ser Asn Lys Lys Lys Arg Asp 195 200 205 Asp Phe Glu
Lys Leu Thr Asn Tyr Ser Val Thr Asp Leu Asn Val Gln 210 215 220 Arg
Lys Ala Ile His Glu Leu Ile Gln Val Met Ala Glu Leu Ser Pro 225 230
235 240 Ala Ala <210> SEQ ID NO 118 <211> LENGTH: 122
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <400> SEQUENCE: 118 Ser Thr Lys Lys Thr Gln Leu
Gln Leu Glu His Leu Leu Leu Asp Leu 1 5 10 15 Gln Met Ile Leu Asn
Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr 20 25 30 Arg Met Leu
Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu 35 40 45 Lys
His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val 50 55
60 Leu Asn Leu Ala Gln Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser
65 70 75 80 Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Phe Met Cys
Glu Tyr 85 90 95 Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn
Arg Trp Ile Thr 100 105 110 Phe Cys Gln Ser Ile Ile Ser Thr Leu Thr
115 120 <210> SEQ ID NO 119 <211> LENGTH: 152
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <400> SEQUENCE: 119 Ala Pro Val Arg Ser Leu Asn
Cys Thr Leu Arg Asp Ser Gln Gln Lys 1 5 10 15 Ser Leu Val Met Ser
Gly Pro Tyr Glu Leu Lys Ala Leu His Leu Gln 20 25 30 Gly Gln Asp
Met Glu Gln Gln Val Val Phe Ser Met Ser Phe Val Gln 35 40 45 Gly
Glu Glu Ser Asn Asp Lys Ile Pro Val Ala Leu Gly Leu Lys Glu 50 55
60 Lys Asn Leu Tyr Leu Ser Cys Val Leu Lys Asp Asp Lys Pro Thr Leu
65 70 75 80 Gln Leu Glu Ser Val Asp Pro Lys Asn Tyr Pro Lys Lys Lys
Met Glu 85 90 95 Lys Arg Phe Val Phe Asn Lys Ile Glu Ile Asn Asn
Lys Leu Glu Phe 100 105 110 Glu Ser Ala Gln Phe Pro Asn Trp Tyr Ile
Ser Thr Ser Gln Ala Glu 115 120 125 Asn Met Pro Val Phe Leu Gly Gly
Thr Lys Gly Gly Gln Asp Ile Thr 130 135 140 Asp Phe Thr Met Gln Phe
Val Ser 145 150 <210> SEQ ID NO 120 <211> LENGTH: 148
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <400> SEQUENCE: 120 Asp Lys Pro Val Ala His Val
Val Ala Asn Pro Gln Ala Glu Gly Gln 1 5 10 15 Leu Gln Trp Ser Asn
Arg Arg Ala Asn Ala Leu Leu Ala Asn Gly Val 20 25 30 Glu Leu Arg
Asp Asn Gln Leu Val Val Pro Ile Glu Gly Leu Phe Leu 35 40 45 Ile
Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly Cys Pro Ser Thr His 50 55
60 Val Leu Leu Thr His Thr Ile Ser Arg Ile Ala Val Ser Tyr Gln Thr
65 70 75 80 Lys Val Asn Leu Leu Ser Ala Ile Lys Ser Pro Cys Gln Arg
Glu Thr 85 90 95 Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu Pro
Ile Tyr Leu Gly 100 105 110 Gly Val Phe Gln Leu Glu Lys Gly Asp Arg
Leu Ser Ala Glu Ile Asn 115 120 125 Arg Pro Asp Tyr Leu Asp Phe Ala
Glu Ser Gly Gln Val Tyr Phe Gly 130 135 140 Ile Ile Ala Leu 145
<210> SEQ ID NO 121 <211> LENGTH: 144 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 121 Lys Pro Ala Ala His Leu Ile Gly Asp Pro Ser Lys Gln
Asn Ser Leu 1 5 10 15 Leu Trp Arg Ala Asn Thr Asp Arg Ala Phe Leu
Gln Asp Gly Phe Ser 20 25 30 Leu Ser Asn Asn Ser Leu Leu Val Pro
Thr Ser Gly Ile Tyr Phe Val 35 40 45 Tyr Ser Gln Val Val Phe Ser
Gly Lys Ala Tyr Ser Pro Lys Ala Thr 50 55 60 Ser Ser Pro Leu Tyr
Leu Ala His Glu Val Gln Leu Phe Ser Ser Gln 65 70 75 80 Tyr Pro Phe
His Val Pro Leu Leu Ser Ser Gln Lys Met Val Tyr Pro 85 90 95 Gly
Leu Gln Glu Pro Trp Leu His Ser Met Tyr His Gly Ala Ala Phe 100 105
110 Gln Leu Thr Gln Gly Asp Gln Leu Ser Thr His Thr Asp Gly Ile Pro
115 120 125 His Leu Val Leu Ser Pro Ser Thr Val Phe Phe Gly Ala Phe
Ala Leu 130 135 140 <210> SEQ ID NO 122 <211> LENGTH:
166 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <400> SEQUENCE: 122 Ala Pro Pro Arg Leu Ile Cys
Asp Ser Arg Val Leu Glu Arg Tyr Leu 1 5 10 15 Leu Glu Ala Lys Glu
Ala Glu Lys Ile Thr Thr Gly Cys Ala Glu His 20 25 30 Cys Ser Leu
Asn Glu Lys Ile Thr Val Pro Asp Thr Lys Val Asn Phe 35 40 45 Tyr
Ala Trp Lys Arg Met Glu Val Gly Gln Gln Ala Val Glu Val Trp 50 55
60 Gln Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu
65 70 75 80 Leu Val Lys Ser Ser Gln Pro Trp Glu Pro Leu Gln Leu His
Val Asp 85 90 95 Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu
Leu Arg Ala Leu 100 105 110 Gly Ala Gln Lys Glu Ala Ile Ser Asn Ser
Asp Ala Ala Ser Ala Ala 115 120 125 Pro Leu Arg Thr Ile Thr Ala Asp
Thr Phe Arg Lys Leu Phe Arg Val 130 135 140 Tyr Ser Asn Phe Leu Arg
Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala 145 150 155 160 Cys Arg Thr
Gly Asp Arg 165 <210> SEQ ID NO 123 <211> LENGTH: 21
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <400> SEQUENCE: 123 Gly Ile Val Glu Gln Cys Cys
Thr Ser Ile Cys Ser Leu Tyr Gln Leu 1 5 10 15 Glu Asn Tyr Cys Asn
20 <210> SEQ ID NO 124 <211> LENGTH: 29 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide
<400> SEQUENCE: 124 Phe Val Asn Gln His Leu Cys Gly Ser His
Leu Val Glu Ala Leu Tyr 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe
Phe Tyr Thr Pro Lys 20 25 <210> SEQ ID NO 125 <211>
LENGTH: 166 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Polypeptide <400> SEQUENCE: 125 Phe Pro Thr Ile Pro
Leu Ser Arg Leu Ala Asp Asn Ala Trp Leu Arg 1 5 10 15 Ala Asp Arg
Leu Asn Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu 20 25 30 Glu
Ala Tyr Ile Pro Lys Glu Gln Ile His Ser Phe Trp Trp Asn Pro 35 40
45 Gln Thr Ser Leu Cys Pro Ser Glu Ser Ile Pro Thr Pro Ser Asn Lys
50 55 60 Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile
Ser Leu 65 70 75 80 Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln Phe
Leu Arg Ser Val 85 90 95 Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser
Asp Ser Asn Val Tyr Asp 100 105 110 Leu Leu Lys Asp Leu Glu Glu Gly
Ile Gln Thr Leu Met Gly Arg Leu 115 120 125 Glu Ala Leu Leu Lys Asn
Tyr Gly Leu Leu Tyr Cys Phe Asn Lys Asp 130 135 140 Met Ser Lys Val
Ser Thr Tyr Leu Arg Thr Val Gln Cys Arg Ser Val 145 150 155 160 Glu
Gly Ser Cys Gly Phe 165 <210> SEQ ID NO 126 <211>
LENGTH: 242 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Polypeptide <400> SEQUENCE: 126 Cys His His Arg Ile
Cys His Cys Ser Asn Arg Val Phe Leu Cys Gln 1 5 10 15 Glu Ser Lys
Val Thr Glu Ile Pro Ser Asp Leu Pro Arg Asn Ala Ile 20 25 30 Glu
Leu Arg Phe Val Leu Thr Lys Leu Arg Val Ile Gln Lys Gly Ala 35 40
45 Phe Ser Gly Phe Gly Asp Leu Glu Lys Ile Glu Ile Ser Gln Asn Asp
50 55 60 Val Leu Glu Val Ile Glu Ala Asp Val Phe Ser Asn Leu Pro
Lys Leu 65 70 75 80 His Glu Ile Arg Ile Glu Lys Ala Asn Asn Leu Leu
Tyr Ile Asn Pro 85 90 95 Glu Ala Phe Gln Asn Leu Pro Asn Leu Gln
Tyr Leu Leu Ile Ser Asn 100 105 110 Thr Gly Ile Lys His Leu Pro Asp
Val His Lys Ile His Ser Leu Gln 115 120 125 Lys Val Leu Leu Asp Ile
Gln Asp Asn Ile Asn Ile His Thr Ile Glu 130 135 140 Arg Asn Ser Phe
Val Gly Leu Ser Phe Glu Ser Val Ile Leu Trp Leu 145 150 155 160 Asn
Lys Asn Gly Ile Gln Glu Ile His Asn Cys Ala Phe Asn Gly Thr 165 170
175 Gln Leu Asp Glu Leu Asn Leu Ser Asp Asn Asn Asn Leu Glu Glu Leu
180 185 190 Pro Asn Asp Val Phe His Gly Ala Ser Gly Pro Val Ile Leu
Asp Ile 195 200 205 Ser Arg Thr Arg Ile His Ser Leu Pro Ser Tyr Gly
Leu Glu Asn Leu 210 215 220 Lys Lys Leu Arg Ala Arg Ser Thr Tyr Asn
Leu Lys Lys Leu Pro Thr 225 230 235 240 Leu Glu <210> SEQ ID
NO 127 <211> LENGTH: 130 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 127
Ile Gln Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr Ile Val 1 5
10 15 Thr Arg Ile Asn Asp Ile Leu Asp Phe Ile Pro Gly Leu His Pro
Ile 20 25 30 Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala Val Tyr
Gln Gln Ile 35 40 45 Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln
Ile Ser Asn Asp Leu 50 55 60 Glu Asn Leu Arg Asp Leu Leu His Val
Leu Ala Phe Ser Lys Ser Cys 65 70 75 80 His Leu Pro Glu Ala Ser Gly
Leu Glu Thr Leu Asp Ser Leu Gly Gly 85 90 95 Val Leu Glu Ala Ser
Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg 100 105 110 Leu Gln Gly
Ser Leu Gln Asp Met Leu Trp Gln Leu Asp Leu Ser Pro 115 120 125 Gly
Cys 130 <210> SEQ ID NO 128 <211> LENGTH: 62
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <400> SEQUENCE: 128 Pro Glu Thr Leu Cys Gly Ala
Glu Leu Val Asp Ala Leu Gln Phe Val 1 5 10 15 Cys Gly Asp Arg Gly
Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly Ser 20 25 30 Ser Ser Arg
Arg Ala Pro Gln Thr Gly Ile Val Asp Glu Cys Cys Phe 35 40 45 Arg
Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys Ala Pro 50 55 60
<210> SEQ ID NO 129 <211> LENGTH: 125 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 129 Met Tyr Arg Ser Ala Phe Ser Val Gly Leu Glu Thr Arg
Val Thr Val 1 5 10 15 Pro Asn Val Pro Ile Arg Phe Thr Lys Ile Phe
Tyr Asn Gln Gln Asn 20 25 30 His Tyr Asp Gly Ser Thr Gly Lys Phe
Tyr Cys Asn Ile Pro Gly Leu 35 40 45 Tyr Tyr Phe Ser Tyr His Ile
Thr Val Tyr Met Lys Asp Val Lys Val 50 55 60 Ser Leu Phe Lys Lys
Asp Lys Ala Val Leu Phe Thr Tyr Asp Gln Tyr 65 70 75 80 Gln Glu Asn
Val Asp Gln Ala Ser Gly Ser Val Leu Leu His Leu Glu 85 90 95 Val
Gly Asp Gln Val Trp Leu Gln Val Tyr Tyr Ala Asp Asn Val Asn 100 105
110 Asp Ser Thr Phe Thr Gly Phe Leu Leu Tyr His Asp Thr 115 120 125
<210> SEQ ID NO 130 <211> LENGTH: 111 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 130 Met Tyr Arg Ser Ala Phe Ser Val Gly Leu Pro Asn Val
Pro Ile Arg 1 5 10 15 Phe Thr Lys Ile Phe Tyr Asn Gln Gln Asn His
Tyr Asp Gly Ser Thr 20 25 30 Gly Lys Phe Tyr Cys Asn Ile Pro Gly
Leu Tyr Tyr Phe Ser Tyr His 35 40 45 Ile Thr Val Tyr Met Lys Asp
Val Lys Val Ser Leu Phe Lys Lys Asp 50 55 60 Lys Val Leu Phe Thr
Tyr Asp Gln Tyr Gln Glu Lys Val Asp Gln Ala 65 70 75 80 Ser Gly Ser
Val Leu Leu His Leu Glu Val Gly Asp Gln Val Trp Leu 85 90 95 Gln
Val Tyr Asp Ser Thr Phe Thr Gly Phe Leu Leu Tyr His Asp 100 105 110
<210> SEQ ID NO 131 <211> LENGTH: 102 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 131 Met Tyr Arg Ser Ala Phe Ser Val Gly Leu Glu Thr Arg
Val Thr Val 1 5 10 15 Pro Ile Arg Phe Thr Lys Ile Phe Tyr Asn Gln
Gln Asn His Tyr Asp 20 25 30 Gly Ser Thr Gly Lys Phe Tyr Cys Asn
Ile Pro Gly Leu Tyr Tyr Phe 35 40 45 Ser Tyr His Ile Thr Val Asp
Val Lys Val Ser Leu Phe Lys Lys Asp 50 55 60 Lys Ala Val Leu Phe
Thr Gln Ala Ser Gly Ser Val Leu Leu His Leu 65 70 75 80 Glu Val Gly
Asp Gln Val Trp Leu Gln Asn Asp Ser Thr Phe Thr Gly 85 90 95 Phe
Leu Leu Tyr His Asp 100 <210> SEQ ID NO 132 <211>
LENGTH: 693 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Polypeptide <400> SEQUENCE: 132 Ala Thr Arg Arg Tyr
Tyr Leu Gly Ala Val Glu Leu Ser Trp Asp Tyr 1 5 10 15 Met Gln Ser
Asp Leu Gly Glu Leu Pro Val Asp Ala Arg Phe Pro Pro 20 25 30 Arg
Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val Tyr Lys Lys 35 40
45 Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile Ala Lys Pro
50 55 60 Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln Ala
Glu Val 65 70 75 80 Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala
Ser His Pro Val 85 90 95 Ser Leu His Ala Val Gly Val Ser Tyr Trp
Lys Ala Ser Glu Gly Ala 100 105 110 Glu Tyr Asp Asp Gln Thr Ser Gln
Arg Glu Lys Glu Asp Asp Lys Val 115 120 125 Phe Pro Gly Gly Ser His
Thr Tyr Val Trp Gln Val Leu Lys Glu Asn 130 135 140 Gly Pro Met Ala
Ser Asp Pro Leu Cys Leu Thr Tyr Ser Tyr Leu Ser 145 150 155 160 His
Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala Leu 165 170
175 Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr Leu
180 185 190 His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly Lys
Ser Trp 195 200 205 His Ser Glu Thr Lys Asn Ala Ala Ser Ala Arg Ala
Trp Pro Lys Met 210 215 220 His Thr Val Asn Gly Tyr Val Asn Arg Ser
Leu Pro Gly Leu Ile Gly 225 230 235 240 Cys His Arg Lys Ser Val Tyr
Trp His Val Ile Gly Met Gly Thr Thr 245 250 255 Pro Glu Val His Ser
Ile Phe Leu Glu Gly His Thr Phe Leu Val Arg 260 265 270 Asn His Arg
Gln Ala Ser Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr 275 280 285 Ala
Gln Thr Leu Leu Met Asp Leu Gly Gln Phe Leu Leu Phe Cys His 290 295
300 Ile Ser Ser His Gln His Asp Gly Met Glu Ala Tyr Val Lys Val Asp
305 310 315 320 Ser Cys Pro Glu Glu Pro Gln Phe Asp Asp Asp Asn Ser
Pro Ser Phe 325 330 335 Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro
Lys Thr Trp Val His 340 345 350 Tyr Ile Ala Ala Glu Glu Glu Asp Trp
Asp Tyr Ala Pro Leu Val Leu 355 360 365 Ala Pro Asp Asp Arg Ser Tyr
Lys Ser Gln Tyr Leu Asn Asn Gly Pro 370 375 380 Gln Arg Ile Gly Arg
Lys Tyr Lys Lys Val Arg Phe Met Ala Tyr Thr 385 390 395 400 Asp Glu
Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu Ser Gly Ile 405 410 415
Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile Ile 420
425 430 Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly
Ile 435 440 445 Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys
Gly Val Lys 450 455 460 His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu
Ile Phe Lys Tyr Lys 465 470 475 480 Trp Thr Val Thr Val Glu Asp Gly
Pro Thr Lys Ser Asp Pro Arg Cys 485 490 495 Leu Thr Arg Tyr Tyr Ser
Ser Phe Val Asn Met Glu Arg Asp Leu Ala 500 505 510 Ser Gly Leu Ile
Gly Pro Leu Leu Ile Cys Tyr Lys Glu Ser Val Asp 515 520 525 Gln Arg
Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val Ile Leu Phe 530 535 540
Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu Asn Ile Gln 545
550 555 560 Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp Pro
Glu Phe 565 570 575 Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr
Val Phe Asp Ser 580 585 590 Leu Gln Leu Ser Val Cys Leu His Glu Val
Ala Tyr Trp Tyr Ile Leu 595 600 605 Ser Ile Gly Ala Gln Thr Asp Phe
Leu Ser Val Phe Phe Ser Gly Tyr 610 615 620 Thr Phe Lys His Lys Met
Val Tyr Glu Asp Thr Leu Thr Leu Phe Pro 625 630 635 640 Phe Ser Gly
Glu Thr Val Phe Met Ser Met Glu Asn Pro Gly Leu Trp 645 650 655 Ile
Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly Met Thr Ala 660 665
670 Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp Tyr Tyr Glu
675 680 685 Asp Ser Tyr Glu Asp 690 <210> SEQ ID NO 133
<211> LENGTH: 644 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 133 Arg
Ser Phe Gln Lys Lys Thr Arg His Tyr Phe Ile Ala Ala Val Glu 1 5 10
15 Arg Leu Trp Asp Tyr Gly Met Ser Ser Ser Pro His Val Leu Arg Asn
20 25 30 Arg Ala Gln Ser Gly Ser Val Pro Gln Phe Lys Lys Val Val
Phe Gln 35 40 45 Glu Phe Thr Asp Gly Ser Phe Thr Gln Pro Leu Tyr
Arg Gly Glu Leu 50 55 60 Asn Glu His Leu Gly Leu Leu Gly Pro Tyr
Ile Arg Ala Glu Val Glu 65 70 75 80 Asp Asn Ile Met Val Thr Phe Arg
Asn Gln Ala Ser Arg Pro Tyr Ser 85 90 95 Phe Tyr Ser Ser Leu Ile
Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala 100 105 110 Glu Pro Arg Lys
Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe 115 120 125 Trp Lys
Val Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp Cys 130 135 140
Lys Ala Trp Ala Tyr Ser Ser Asp Val Asp Leu Glu Lys Asp Val His 145
150 155 160 Ser Gly Leu Ile Gly Pro Leu Leu Val Cys His Thr Asn Thr
Leu Asn 165 170 175 Pro Ala His Gly Arg Gln Val Thr Val Gln Glu Phe
Ala Leu Phe Phe 180 185 190 Thr Ile Phe Asp Glu Thr Lys Ser Trp Tyr
Phe Thr Glu Asn Met Glu 195 200 205 Arg Asn Cys Arg Ala Pro Cys Asn
Ile Gln Met Glu Asp Pro Thr Phe 210 215 220 Lys Glu Asn Tyr Arg Phe
His Ala Ile Asn Gly Tyr Ile Met Asp Thr 225 230 235 240 Leu Pro Gly
Leu Val Met Ala Gln Asp Gln Arg Ile Arg Trp Tyr Leu 245 250 255 Leu
Ser Met Gly Ser Asn Glu Asn Ile His Ser Ile His Phe Ser Gly 260 265
270 His Val Phe Thr Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Leu Tyr
275 280 285 Asn Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro
Ser Lys 290 295 300 Ala Gly Ile Trp Arg Val Glu Cys Leu Ile Gly Glu
His Leu His Ala 305 310 315 320 Gly Met Ser Thr Leu Phe Leu Val Tyr
Ser Asn Lys Cys Gln Thr Pro 325 330 335 Leu Gly Met Ala Ser Gly His
Ile Arg Asp Phe Gln Ile Thr Ala Ser 340 345 350 Gly Gln Tyr Gly Gln
Trp Ala Pro Lys Leu Ala Arg Leu His Tyr Ser 355 360 365 Gly Ser Ile
Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser Trp Ile Lys 370 375 380 Val
Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile Lys Thr Gln Gly 385 390
395 400 Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile
Met 405 410 415 Tyr Ser Leu Asp Gly Lys Lys Trp Gln Thr Tyr Arg Gly
Asn Ser Thr 420 425 430 Gly Thr Leu Met Val Phe Phe Gly Asn Val Asp
Ser Ser Gly Ile Lys 435 440 445 His Asn Ile Phe Asn Pro Pro Ile Ile
Ala Arg Tyr Ile Arg Leu His 450 455 460 Pro Thr His Tyr Ser Ile Arg
Ser Thr Leu Arg Met Glu Leu Met Gly 465 470 475 480 Cys Asp Leu Asn
Ser Cys Ser Met Pro Leu Gly Met Glu Ser Lys Ala 485 490 495 Ile Ser
Asp Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr Asn Met Phe 500 505 510
Ala Thr Trp Ser Pro Ser Lys Ala Arg Leu His Leu Gln Gly Arg Ser 515
520 525 Asn Ala Trp Arg Pro Gln Val Asn Asn Pro Lys Glu Trp Leu Gln
Val 530 535 540 Asp Phe Gln Lys Thr Met Lys Val Thr Gly Val Thr Thr
Gln Gly Val 545 550 555 560 Lys Ser Leu Leu Thr Ser Met Tyr Val Lys
Glu Phe Leu Ile Ser Ser 565 570 575 Ser Gln Asp Gly His Gln Trp Thr
Leu Phe Phe Gln Asn Gly Lys Val 580 585 590 Lys Val Phe Gln Gly Asn
Gln Asp Ser Phe Thr Pro Val Val Asn Ser 595 600 605 Leu Asp Pro Pro
Leu Leu Thr Arg Tyr Leu Arg Ile His Pro Gln Ser 610 615 620 Trp Val
His Gln Ile Ala Leu Arg Met Glu Val Leu Gly Cys Glu Ala 625 630 635
640 Gln Asp Leu Tyr <210> SEQ ID NO 134 <211> LENGTH:
578 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <400> SEQUENCE: 134 Ser Glu Val Ala His Arg Phe
Lys Asp Leu Gly Glu Glu Asn Phe Lys 1 5 10 15 Ala Leu Val Leu Ile
Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe 20 25 30 Glu Asp His
Val Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr 35 40 45 Cys
Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr 50 55
60 Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr
65 70 75 80 Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg
Asn Glu 85 90 95 Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu
Pro Arg Leu Val 100 105 110 Arg Pro Glu Val Asp Val Met Cys Thr Ala
Phe His Asp Asn Glu Glu 115 120 125 Thr Phe Leu Lys Lys Tyr Leu Tyr
Glu Ile Ala Arg Arg His Pro Tyr 130 135 140 Phe Tyr Ala Pro Glu Leu
Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala 145 150 155 160 Phe Thr Glu
Cys Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro 165 170 175 Lys
Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln 180 185
190 Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys
195 200 205 Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala
Glu Phe 210 215 220 Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys
Val His Thr Glu 225 230 235 240 Cys Cys His Gly Asp Leu Leu Glu Cys
Ala Asp Asp Arg Ala Asp Leu 245 250 255 Ala Lys Tyr Ile Cys Glu Asn
Gln Asp Ser Ile Ser Ser Lys Leu Lys 260 265 270 Glu Cys Cys Glu Lys
Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu 275 280 285 Val Glu Asn
Asp Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp 290 295 300 Phe
Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp 305 310
315 320 Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro
Asp 325 330 335 Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr Tyr
Glu Thr Thr 340 345 350 Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His
Glu Cys Tyr Ala Lys 355 360 365 Val Phe Asp Glu Phe Lys Pro Leu Val
Glu Glu Pro Gln Asn Leu Ile 370 375 380 Lys Gln Asn Cys Glu Leu Phe
Glu Gln Leu Gly Glu Tyr Lys Phe Gln 385 390 395 400 Asn Ala Leu Leu
Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr 405 410 415 Pro Thr
Leu Val Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys 420 425 430
Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr 435
440 445 Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr
Pro 450 455 460 Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser Leu
Val Asn Arg 465 470 475 480 Arg Pro Cys Phe Ser Ala Leu Glu Val Asp
Glu Thr Tyr Val Pro Lys 485 490 495 Glu Phe Asn Ala Glu Thr Phe Thr
Phe His Ala Asp Ile Cys Thr Leu 500 505 510 Ser Glu Lys Glu Arg Gln
Ile Lys Lys Gln Thr Ala Leu Val Glu Leu 515 520 525 Val Lys His Lys
Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met 530 535 540 Asp Asp
Phe Ala Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys 545 550 555
560 Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln
565 570 575 Ala Ala <210> SEQ ID NO 135 <211> LENGTH:
578 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <400> SEQUENCE: 135 Ser Glu Val Ala His Arg Phe
Lys Asp Leu Gly Glu Glu Asn Phe Lys 1 5 10 15 Ala Leu Val Leu Ile
Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe 20 25 30 Glu Asp His
Val Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr 35 40 45 Cys
Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr 50 55
60 Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr
65 70 75 80 Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg
Asn Glu 85 90 95 Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu
Pro Arg Leu Val 100 105 110 Arg Pro Glu Val Asp Val Met Cys Thr Ala
Phe His Asp Asn Glu Glu 115 120 125 Thr Phe Leu Lys Lys Tyr Leu Tyr
Glu Ile Ala Arg Arg His Pro Tyr 130 135 140 Phe Tyr Ala Pro Glu Leu
Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala 145 150 155 160 Phe Thr Glu
Cys Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro 165 170 175 Lys
Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln 180 185
190 Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys
195 200 205 Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala
Glu Phe 210 215 220 Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys
Val His Thr Glu 225 230 235 240 Cys Cys His Gly Asp Leu Leu Glu Cys
Ala Asp Asp Arg Ala Asp Leu 245 250 255 Ala Lys Tyr Ile Cys Glu Asn
Gln Asp Ser Ile Ser Ser Lys Leu Lys 260 265 270 Glu Cys Cys Glu Lys
Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu 275 280 285 Val Glu Asn
Asp Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp 290 295 300 Phe
Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp 305 310
315 320 Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro
Asp 325 330 335 Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr Tyr
Glu Thr Thr 340 345 350 Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His
Glu Cys Tyr Ala Lys 355 360 365 Val Phe Asp Glu Phe Lys Pro Leu Val
Glu Glu Pro Gln Asn Leu Ile 370 375 380 Lys Gln Asn Cys Glu Leu Phe
Glu Gln Leu Gly Glu Tyr Lys Phe Gln 385 390 395 400 Asn Ala Leu Leu
Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr 405 410 415 Pro Thr
Leu Val Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys 420 425 430
Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr 435
440 445 Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr
Pro 450 455 460 Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser Leu
Val Asn Arg 465 470 475 480 Arg Pro Cys Phe Ser Ala Leu Glu Val Asp
Glu Thr Tyr Val Pro Lys 485 490 495 Glu Phe Asn Ala Glu Thr Phe Thr
Phe His Ala Asp Ile Cys Thr Leu 500 505 510 Ser Glu Lys Glu Arg Gln
Ile Lys Lys Gln Thr Ala Leu Val Glu Leu 515 520 525 Val Lys His Lys
Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met 530 535 540 Asp Asp
Phe Ala Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys 545 550 555
560 Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln
565 570 575 Ala Ala <210> SEQ ID NO 136 <211> LENGTH:
141 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <400> SEQUENCE: 136 Val Leu Ser Pro Ala Asp Lys
Thr Asn Val Lys Ala Ala Trp Gly Lys 1 5 10 15 Val Gly Ala His Ala
Gly Glu Tyr Gly Ala Glu Ala Leu Glu Arg Met 20 25 30 Phe Leu Ser
Phe Pro Thr Thr Lys Thr Tyr Phe Pro His Phe Asp Leu 35 40 45 Ser
His Gly Ser Ala Gln Val Lys Gly His Gly Lys Lys Val Ala Asp 50 55
60 Ala Leu Thr Asn Ala Val Ala His Val Asp Asp Met Pro Asn Ala Leu
65 70 75 80 Ser Ala Leu Ser Asp Leu His Ala His Lys Leu Arg Val Asp
Pro Val 85 90 95 Asn Phe Lys Leu Leu Ser His Cys Leu Leu Val Thr
Leu Ala Ala His 100 105 110 Leu Pro Ala Glu Phe Thr Pro Ala Val His
Ala Ser Leu Asp Lys Phe 115 120 125 Leu Ala Ser Val Ser Thr Val Leu
Thr Ser Lys Tyr Arg 130 135 140 <210> SEQ ID NO 137
<211> LENGTH: 146 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 137 Val
His Leu Thr Pro Glu Glu Lys Ser Ala Val Thr Ala Leu Trp Gly 1 5 10
15 Lys Val Asn Val Asp Glu Val Gly Gly Glu Ala Leu Gly Arg Leu Leu
20 25 30 Val Val Tyr Pro Trp Thr Gln Arg Phe Phe Glu Ser Phe Gly
Asp Leu 35 40 45 Ser Thr Pro Asp Ala Val Met Gly Asn Pro Lys Val
Lys Ala His Gly 50 55 60 Lys Lys Val Leu Gly Ala Phe Ser Asp Gly
Leu Ala His Leu Asp Asn 65 70 75 80 Leu Lys Gly Thr Phe Ala Thr Leu
Ser Glu Leu His Cys Asp Lys Leu 85 90 95 His Val Asp Pro Glu Asn
Phe Arg Leu Leu Gly Asn Val Leu Val Cys 100 105 110 Val Leu Ala His
His Phe Gly Lys Glu Phe Thr Pro Pro Val Gln Ala 115 120 125 Ala Tyr
Gln Lys Val Val Ala Gly Val Ala Asn Ala Leu Ala His Lys 130 135 140
Tyr His 145 <210> SEQ ID NO 138 <211> LENGTH: 7
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <400> SEQUENCE: 138 His Ala Leu Pro Glu Thr Gly 1
5
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 138
<210> SEQ ID NO 1 <211> LENGTH: 206 <212> TYPE:
PRT <213> ORGANISM: S. aureus <400> SEQUENCE: 1 Met Lys
Lys Trp Thr Asn Arg Leu Met Thr Ile Ala Gly Val Val Leu 1 5 10 15
Ile Leu Val Ala Ala Tyr Leu Phe Ala Lys Pro His Ile Asp Asn Tyr 20
25 30 Leu His Asp Lys Asp Lys Asp Glu Lys Ile Glu Gln Tyr Asp Lys
Asn 35 40 45 Val Lys Glu Gln Ala Ser Lys Asp Asn Lys Gln Gln Ala
Lys Pro Gln 50 55 60 Ile Pro Lys Asp Lys Ser Lys Val Ala Gly Tyr
Ile Glu Ile Pro Asp 65 70 75 80 Ala Asp Ile Lys Glu Pro Val Tyr Pro
Gly Pro Ala Thr Pro Glu Gln 85 90 95 Leu Asn Arg Gly Val Ser Phe
Ala Glu Glu Asn Glu Ser Leu Asp Asp 100 105 110 Gln Asn Ile Ser Ile
Ala Gly His Thr Phe Ile Asp Arg Pro Asn Tyr 115 120 125 Gln Phe Thr
Asn Leu Lys Ala Ala Lys Lys Gly Ser Met Val Tyr Phe 130 135 140 Lys
Val Gly Asn Glu Thr Arg Lys Tyr Lys Met Thr Ser Ile Arg Asp 145 150
155 160 Val Lys Pro Thr Asp Val Glu Val Leu Asp Glu Gln Lys Gly Lys
Asp 165 170 175 Lys Gln Leu Thr Leu Ile Thr Cys Asp Asp Tyr Asn Glu
Lys Thr Gly 180 185 190 Val Trp Glu Lys Arg Lys Ile Phe Val Ala Thr
Glu Val Lys 195 200 205 <210> SEQ ID NO 2 <211> LENGTH:
206 <212> TYPE: PRT <213> ORGANISM: S. aureus
<400> SEQUENCE: 2 Met Lys Lys Trp Thr Asn Arg Leu Met Thr Ile
Ala Gly Val Val Leu 1 5 10 15 Ile Leu Val Ala Ala Tyr Leu Phe Ala
Lys Pro His Ile Asp Asn Tyr 20 25 30 Leu His Asp Lys Asp Lys Asp
Glu Lys Ile Glu Gln Tyr Asp Lys Asn 35 40 45 Val Lys Glu Gln Ala
Ser Lys Asp Lys Lys Gln Gln Ala Lys Pro Gln 50 55 60 Ile Pro Lys
Asp Lys Ser Lys Val Ala Gly Tyr Ile Glu Ile Pro Asp 65 70 75 80 Ala
Asp Ile Lys Glu Pro Val Tyr Pro Gly Pro Ala Thr Pro Glu Gln 85 90
95 Leu Asn Arg Gly Val Ser Phe Ala Glu Glu Asn Glu Ser Leu Asp Asp
100 105 110 Gln Asn Ile Ser Ile Ala Gly His Thr Phe Ile Asp Arg Pro
Asn Tyr 115 120 125 Gln Phe Thr Asn Leu Lys Ala Ala Lys Lys Gly Ser
Met Val Tyr Phe 130 135 140 Lys Val Gly Asn Glu Thr Arg Lys Tyr Lys
Met Thr Ser Ile Arg Asp 145 150 155 160 Val Lys Pro Thr Asp Val Gly
Val Leu Asp Glu Gln Lys Gly Lys Asp 165 170 175 Lys Gln Leu Thr Leu
Ile Thr Cys Asp Asp Tyr Asn Glu Lys Thr Gly 180 185 190 Val Trp Glu
Lys Arg Lys Ile Phe Val Ala Thr Glu Val Lys 195 200 205 <210>
SEQ ID NO 3 <211> LENGTH: 148 <212> TYPE: PRT
<213> ORGANISM: S. aureus <400> SEQUENCE: 3 Met Gln Ala
Lys Pro Gln Ile Pro Lys Asp Lys Ser Lys Val Ala Gly 1 5 10 15 Tyr
Ile Glu Ile Pro Asp Ala Asp Ile Lys Glu Pro Val Tyr Pro Gly 20 25
30 Pro Ala Thr Arg Glu Gln Leu Asn Arg Gly Val Ser Phe Ala Glu Glu
35 40 45 Asn Glu Ser Leu Asp Asp Gln Asn Ile Ser Ile Ala Gly His
Thr Phe 50 55 60 Ile Asp Arg Pro Asn Tyr Gln Phe Thr Asn Leu Lys
Ala Ala Lys Lys 65 70 75 80 Gly Ser Met Val Tyr Phe Lys Val Gly Asn
Glu Thr Arg Lys Tyr Lys 85 90 95 Met Thr Ser Ile Arg Asn Val Lys
Pro Thr Ala Val Glu Val Leu Asp 100 105 110 Glu Gln Lys Gly Lys Asp
Lys Gln Leu Thr Leu Ile Thr Cys Asp Asp 115 120 125 Tyr Asn Glu Glu
Thr Gly Val Trp Glu Thr Arg Lys Ile Phe Val Ala 130 135 140 Thr Glu
Val Lys 145 <210> SEQ ID NO 4 <211> LENGTH: 148
<212> TYPE: PRT <213> ORGANISM: S. aureus <400>
SEQUENCE: 4 Met Gln Ala Lys Pro Gln Ile Pro Lys Asp Lys Ser Lys Val
Ala Gly 1 5 10 15 Tyr Ile Glu Ile Pro Asp Ala Asp Ile Lys Glu Pro
Val Tyr Pro Gly 20 25 30 Pro Ala Thr Arg Glu Gln Leu Asn Arg Gly
Val Ser Phe Ala Lys Glu 35 40 45 Asn Gln Ser Leu Asp Asp Gln Asn
Ile Ser Ile Ala Gly His Thr Phe 50 55 60 Ile Asp Arg Pro Asn Tyr
Gln Phe Thr Asn Leu Lys Ala Ala Lys Lys 65 70 75 80 Gly Ser Met Val
Tyr Phe Lys Val Gly Asn Glu Thr Arg Lys Tyr Lys 85 90 95 Met Thr
Ser Ile Arg Asn Val Lys Pro Thr Ala Val Glu Val Leu Asp 100 105 110
Glu Gln Lys Gly Lys Asp Lys Gln Leu Thr Leu Ile Thr Cys Asp Asp 115
120 125 Tyr Asn Glu Glu Thr Gly Val Trp Glu Thr Arg Lys Ile Phe Val
Ala 130 135 140 Thr Glu Val Lys 145 <210> SEQ ID NO 5
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Polypeptide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (2)..(2) <223>
OTHER INFORMATION: Xaa is Val or Ile <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (4)..(4)
<223> OTHER INFORMATION: Xaa can be any naturally occurring
amino acid <400> SEQUENCE: 5 Asp Xaa Glu Xaa Asn Pro Gly 1 5
<210> SEQ ID NO 6 <211> LENGTH: 22 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 6 Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly
Asp Val 1 5 10 15 Glu Ser Asn Pro Gly Pro 20 <210> SEQ ID NO
7 <211> LENGTH: 156 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 7
Met Gln Ala Lys Pro Gln Ile Pro Lys Asp Lys Ser Lys Val Ala Gly 1 5
10 15 Tyr Ile Glu Ile Pro Asp Ala Asp Ile Lys Glu Pro Val Tyr Pro
Gly 20 25 30 Pro Ala Thr Arg Glu Gln Leu Asn Arg Gly Val Ser Phe
Ala Lys Glu 35 40 45 Asn Gln Ser Leu Asp Asp Gln Asn Ile Ser Ile
Ala Gly His Thr Phe 50 55 60 Ile Asp Arg Pro Asn Tyr Gln Phe Thr
Asn Leu Lys Ala Ala Lys Lys 65 70 75 80 Gly Ser Met Val Tyr Phe Lys
Val Gly Asn Glu Thr Arg Lys Tyr Lys 85 90 95 Met Thr Ser Ile Arg
Asn Val Lys Pro Thr Ala Val Glu Val Leu Asp 100 105 110 Glu Gln Lys
Gly Lys Asp Lys Gln Leu Thr Leu Ile Thr Cys Asp Asp 115 120 125 Tyr
Asn Glu Glu Thr Gly Val Trp Glu Thr Arg Lys Ile Phe Val Ala 130 135
140
Thr Glu Val Lys Leu Glu His His His His His His 145 150 155
<210> SEQ ID NO 8 <211> LENGTH: 5 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(3)..(3) <223> OTHER INFORMATION: Xaa can be any naturally
occurring amino acid <400> SEQUENCE: 8 Leu Pro Xaa Thr Gly 1
5 <210> SEQ ID NO 9 <211> LENGTH: 6 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(3)..(3) <223> OTHER INFORMATION: Xaa can be any naturally
occurring amino acid <400> SEQUENCE: 9 Leu Pro Xaa Thr Gly
Gly 1 5 <210> SEQ ID NO 10 <211> LENGTH: 6 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide
<400> SEQUENCE: 10 Leu Pro Glu Thr Gly Gly 1 5 <210>
SEQ ID NO 11 <211> LENGTH: 6 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(4)..(4) <223> OTHER INFORMATION: Xaa can be any naturally
occurring amino acid <400> SEQUENCE: 11 Lys Leu Pro Xaa Thr
Gly 1 5 <210> SEQ ID NO 12 <211> LENGTH: 5 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide
<400> SEQUENCE: 12 Leu Pro Glu Thr Gly 1 5 <210> SEQ ID
NO 13 <211> LENGTH: 8 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 13
Ser Ile Ile Asn Phe Glu Lys Leu 1 5 <210> SEQ ID NO 14
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 14 Leu Pro
Glu Thr Gly 1 5 <210> SEQ ID NO 15 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <400> SEQUENCE: 15 Leu Pro Glu Thr Gly 1 5
<210> SEQ ID NO 16 <211> LENGTH: 4 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(4)..(4) <223> OTHER INFORMATION: Ser can be replaced by Thr,
Gly or Ala <400> SEQUENCE: 16 Gly Gly Gly Ser 1 <210>
SEQ ID NO 17 <211> LENGTH: 5 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 17 Gly Gly Gly Gly Ser 1 5 <210> SEQ ID NO 18
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Polypeptide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (2)..(2) <223>
OTHER INFORMATION: Xaa can be any naturally occurring amino acid
<400> SEQUENCE: 18 Leu Xaa Pro Thr Gly 1 5 <210> SEQ ID
NO 19 <211> LENGTH: 6 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 19
His His His His His His 1 5 <210> SEQ ID NO 20 <400>
SEQUENCE: 20 000 <210> SEQ ID NO 21 <400> SEQUENCE: 21
000 <210> SEQ ID NO 22 <211> LENGTH: 4 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide
<400> SEQUENCE: 22 Gly Gly Gly Gly 1 <210> SEQ ID NO 23
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 23 Gly Gly
Gly Gly Gly 1 5 <210> SEQ ID NO 24 <211> LENGTH: 4
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <400> SEQUENCE: 24 Ala Ala Ala Ala 1 <210>
SEQ ID NO 25 <211> LENGTH: 5 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 25 Ala Ala Ala Ala Ala 1 5 <210> SEQ ID NO 26
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 26 Leu Pro
Lys Thr Gly 1 5 <210> SEQ ID NO 27 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <400> SEQUENCE: 27 Leu Pro Ala Thr Gly 1 5
<210> SEQ ID NO 28 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 28 Leu Pro Asn Thr Gly 1 5 <210> SEQ ID NO 29
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Polypeptide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (3)..(3) <223>
OTHER INFORMATION: Xaa can be any naturally occurring amino acid
<400> SEQUENCE: 29 Leu Pro Xaa Ala Gly 1 5 <210> SEQ ID
NO 30 <211> LENGTH: 5 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 30
Leu Pro Asn Ala Gly 1 5 <210> SEQ ID NO 31 <211>
LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Polypeptide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (3)..(3) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid
<400> SEQUENCE: 31 Leu Pro Xaa Thr Ala 1 5 <210> SEQ ID
NO 32 <211> LENGTH: 5 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 32
Leu Pro Asn Thr Ala 1 5 <210> SEQ ID NO 33 <211>
LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Polypeptide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (3)..(3) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid
<400> SEQUENCE: 33 Leu Gly Xaa Thr Gly 1 5 <210> SEQ ID
NO 34 <211> LENGTH: 5 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 34
Leu Gly Ala Thr Gly 1 5 <210> SEQ ID NO 35 <211>
LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Polypeptide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (3)..(3) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid
<400> SEQUENCE: 35 Ile Pro Xaa Thr Gly 1 5 <210> SEQ ID
NO 36 <211> LENGTH: 5 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 36
Ile Pro Asn Thr Gly 1 5 <210> SEQ ID NO 37 <211>
LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Polypeptide <400> SEQUENCE: 37 Ile Pro Glu Thr Gly
1 5 <210> SEQ ID NO 38 <211> LENGTH: 5 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (3)..(3) <223> OTHER INFORMATION: Xaa can be any
naturally occurring amino acid <400> SEQUENCE: 38 Thr Leu Xaa
Thr Cys 1 5 <210> SEQ ID NO 39 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (3)..(3) <223> OTHER INFORMATION: Xaa
is Gln or Lys <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(4) <223> OTHER
INFORMATION: Xaa is Thr or Ser <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (5)..(5) <223>
OTHER INFORMATION: Xaa is Asn, Gly or Ser <400> SEQUENCE: 39
Asn Pro Xaa Xaa Xaa 1 5 <210> SEQ ID NO 40 <211>
LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Polypeptide <400> SEQUENCE: 40 Asn Pro Gln Thr Asn
1 5 <210> SEQ ID NO 41 <211> LENGTH: 5 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide
<400> SEQUENCE: 41 Asn Pro Lys Thr Gly 1 5 <210> SEQ ID
NO 42 <211> LENGTH: 5 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Polypeptide
<400> SEQUENCE: 42 Asn Ser Lys Thr Ala 1 5 <210> SEQ ID
NO 43 <211> LENGTH: 5 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 43
Asn Pro Gln Thr Gly 1 5 <210> SEQ ID NO 44 <211>
LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Polypeptide <400> SEQUENCE: 44 Asn Ala Lys Thr Asn
1 5 <210> SEQ ID NO 45 <211> LENGTH: 5 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide
<400> SEQUENCE: 45 Asn Pro Gln Ser Ser 1 5 <210> SEQ ID
NO 46 <211> LENGTH: 5 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Polypeptide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (3)..(3)
<223> OTHER INFORMATION: Xaa can be any naturally occurring
amino acid <400> SEQUENCE: 46 Asn Ala Xaa Thr Gly 1 5
<210> SEQ ID NO 47 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(3)..(3) <223> OTHER INFORMATION: Xaa can be any naturally
occurring amino acid <400> SEQUENCE: 47 Leu Ala Xaa Thr Gly 1
5 <210> SEQ ID NO 48 <211> LENGTH: 6 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 48 Gln Val Pro Thr Gly Val 1 5 <210> SEQ ID NO 49
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Polypeptide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (3)..(3) <223>
OTHER INFORMATION: Xaa can be any naturally occurring amino acid
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (5)..(5) <223> OTHER INFORMATION: Xaa is Ala or Ser
<400> SEQUENCE: 49 Leu Pro Xaa Thr Xaa 1 5 <210> SEQ ID
NO 50 <211> LENGTH: 5 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 50
Leu Pro Ser Thr Ser 1 5 <210> SEQ ID NO 51 <211>
LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Polypeptide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (3)..(3) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (5)..(5) <223> OTHER INFORMATION: Xaa can be any
naturally occurring amino acid <400> SEQUENCE: 51 Ser Pro Xaa
Thr Xaa 1 5 <210> SEQ ID NO 52 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (3)..(3) <223> OTHER INFORMATION: Xaa
can be any naturally occurring amino acid <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (5)..(5)
<223> OTHER INFORMATION: Xaa can be any naturally occurring
amino acid <400> SEQUENCE: 52 Leu Ser Xaa Thr Xaa 1 5
<210> SEQ ID NO 53 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(3)..(3) <223> OTHER INFORMATION: Xaa is Asp, Glu, Ala, Asn,
Gln, Lys or Arg <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (5)..(5) <223> OTHER
INFORMATION: Xaa is Gly or Ser <400> SEQUENCE: 53 Asn Pro Xaa
Thr Xaa 1 5 <210> SEQ ID NO 54 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (3)..(3) <223> OTHER INFORMATION: Xaa
is Asp, Glu, Ala, Asn, Gln, Lys or Arg <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (5)..(5)
<223> OTHER INFORMATION: Xaa is Gly or Ser <400>
SEQUENCE: 54 Val Pro Xaa Thr Xaa 1 5 <210> SEQ ID NO 55
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Polypeptide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (3)..(3) <223>
OTHER INFORMATION: Xaa is Asp, Glu, Ala, Asn, Gln, Lys or Arg
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (5)..(5) <223> OTHER INFORMATION: Xaa is Gly or Ser
<400> SEQUENCE: 55 Ile Pro Xaa Thr Xaa 1 5 <210> SEQ ID
NO 56 <211> LENGTH: 5 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Polypeptide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (3)..(3)
<223> OTHER INFORMATION: Xaa is Asp, Glu, Ala, Asn, Gln, Lys
or Arg <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (5)..(5)
<223> OTHER INFORMATION: Xaa is Gly or Ser <400>
SEQUENCE: 56 Tyr Pro Xaa Arg Xaa 1 5 <210> SEQ ID NO 57
<211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 57 Leu Pro
Lys Thr 1 <210> SEQ ID NO 58 <211> LENGTH: 4
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <400> SEQUENCE: 58 Leu Pro Ile Thr 1 <210>
SEQ ID NO 59 <211> LENGTH: 4 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 59 Leu Pro Asp Thr 1 <210> SEQ ID NO 60 <211>
LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Polypeptide <400> SEQUENCE: 60 Ser Pro Lys Thr 1
<210> SEQ ID NO 61 <211> LENGTH: 4 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 61 Leu Ala Glu Thr 1 <210> SEQ ID NO 62 <211>
LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Polypeptide <400> SEQUENCE: 62 Leu Ala Ala Thr 1
<210> SEQ ID NO 63 <211> LENGTH: 4 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 63 Leu Ala Ser Thr 1 <210> SEQ ID NO 64 <211>
LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Polypeptide <400> SEQUENCE: 64 Leu Pro Leu Thr 1
<210> SEQ ID NO 65 <211> LENGTH: 4 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 65 Leu Ser Arg Thr 1 <210> SEQ ID NO 66 <211>
LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Polypeptide <400> SEQUENCE: 66 Leu Pro Glu Thr 1
<210> SEQ ID NO 67 <211> LENGTH: 4 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 67 Val Pro Asp Thr 1 <210> SEQ ID NO 68 <211>
LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Polypeptide <400> SEQUENCE: 68 Ile Pro Gln Thr 1
<210> SEQ ID NO 69 <211> LENGTH: 4 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 69 Tyr Pro Arg Arg 1 <210> SEQ ID NO 70 <211>
LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Polypeptide <400> SEQUENCE: 70 Leu Pro Met Thr 1
<210> SEQ ID NO 71 <211> LENGTH: 4 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 71 Leu Ala Phe Thr 1 <210> SEQ ID NO 72 <211>
LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Polypeptide <400> SEQUENCE: 72 Leu Pro Gln Thr 1
<210> SEQ ID NO 73 <211> LENGTH: 4 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 73 Asn Ser Lys Thr 1 <210> SEQ ID NO 74 <211>
LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Polypeptide <400> SEQUENCE: 74 Asn Pro Gln Thr 1
<210> SEQ ID NO 75 <211> LENGTH: 4 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 75
Asn Ala Lys Thr 1 <210> SEQ ID NO 76 <211> LENGTH: 4
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <400> SEQUENCE: 76 Asn Pro Gln Ser 1 <210>
SEQ ID NO 77 <211> LENGTH: 5 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 77 Leu Pro Lys Thr Gly 1 5 <210> SEQ ID NO 78
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 78 Leu Pro
Ile Thr Gly 1 5 <210> SEQ ID NO 79 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <400> SEQUENCE: 79 Leu Pro Asp Thr Ala 1 5
<210> SEQ ID NO 80 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 80 Ser Pro Lys Thr Gly 1 5 <210> SEQ ID NO 81
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 81 Leu Ala
Glu Thr Gly 1 5 <210> SEQ ID NO 82 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <400> SEQUENCE: 82 Leu Ala Ala Thr Gly 1 5
<210> SEQ ID NO 83 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 83 Leu Ala His Thr Gly 1 5 <210> SEQ ID NO 84
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 84 Leu Ala
Ser Thr Gly 1 5 <210> SEQ ID NO 85 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <400> SEQUENCE: 85 Leu Ala Glu Thr Gly 1 5
<210> SEQ ID NO 86 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 86 Leu Pro Leu Thr Gly 1 5 <210> SEQ ID NO 87
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 87 Leu Ser
Arg Thr Gly 1 5 <210> SEQ ID NO 88 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <400> SEQUENCE: 88 Leu Pro Glu Thr Gly 1 5
<210> SEQ ID NO 89 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 89 Val Pro Asp Thr Gly 1 5 <210> SEQ ID NO 90
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 90 Ile Pro
Gln Thr Gly 1 5 <210> SEQ ID NO 91 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <400> SEQUENCE: 91 Tyr Pro Arg Arg Gly 1 5
<210> SEQ ID NO 92 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 92 Leu Pro Met Thr Gly 1 5 <210> SEQ ID NO 93
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 93 Leu Pro
Leu Thr Gly 1 5 <210> SEQ ID NO 94 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <400> SEQUENCE: 94 Leu Ala Phe Thr Gly 1 5
<210> SEQ ID NO 95 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 95 Leu Pro Gln Thr Ser 1 5 <210> SEQ ID NO 96
<211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 96 Leu Pro
Ser Thr 1 <210> SEQ ID NO 97 <211> LENGTH: 4
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <400> SEQUENCE: 97 Arg Ala Lys Arg 1 <210>
SEQ ID NO 98 <211> LENGTH: 9 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 98 Thr Thr Cys Cys Gly Leu Arg Gln Tyr 1 5 <210>
SEQ ID NO 99 <211> LENGTH: 303 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 99 Ile Lys Gly Gly Leu Phe Ala Asp Ile Ala Ser His Pro
Trp Gln Ala 1 5 10 15 Ala Ile Phe Ala Lys His His Arg Arg Gly Gly
Glu Arg Phe Leu Cys 20 25 30 Gly Gly Ile Leu Ile Ser Ser Cys Trp
Ile Leu Ser Ala Ala His Cys 35 40 45 Phe Gln Gln Gln Gln Gln Glu
Glu Glu Glu Glu Arg Arg Arg Arg Arg 50 55 60 Phe Phe Phe Phe Phe
Pro Pro Pro Pro Pro Pro His His Leu Thr Val 65 70 75 80 Ile Leu Gly
Arg Thr Tyr Arg Val Val Pro Gly Glu Glu Glu Gln Lys 85 90 95 Phe
Glu Val Glu Lys Tyr Ile Val His Lys Glu Phe Asp Asp Asp Thr 100 105
110 Tyr Asp Asn Asp Ile Ala Leu Leu Gln Leu Lys Ser Ser Ser Ser Ser
115 120 125 Asp Asp Asp Asp Asp Ser Ser Ser Ser Ser Ser Ser Ser Ser
Ser Arg 130 135 140 Arg Arg Arg Arg Cys Ala Gln Glu Ser Ser Val Val
Arg Thr Val Cys 145 150 155 160 Leu Pro Pro Ala Asp Leu Gln Leu Pro
Asp Trp Thr Glu Cys Glu Leu 165 170 175 Ser Gly Tyr Gly Lys His Glu
Ala Leu Ser Pro Phe Tyr Ser Glu Arg 180 185 190 Leu Lys Glu Ala His
Val Arg Leu Tyr Pro Ser Ser Arg Cys Thr Thr 195 200 205 Thr Ser Ser
Ser Gln Gln Gln His Leu Leu Asn Arg Thr Val Thr Asp 210 215 220 Asn
Met Leu Cys Ala Gly Asp Thr Thr Thr Arg Arg Arg Ser Ser Ser 225 230
235 240 Asn Asn Asn Leu His Asp Ala Cys Gln Gly Asp Ser Gly Gly Pro
Leu 245 250 255 Val Cys Leu Asn Asp Gly Arg Met Thr Leu Val Gly Ile
Ile Ser Trp 260 265 270 Gly Leu Gly Cys Gly Gly Gln Gln Lys Asp Val
Pro Gly Val Tyr Thr 275 280 285 Lys Val Thr Asn Tyr Leu Asp Trp Ile
Arg Asp Asn Met Arg Pro 290 295 300 <210> SEQ ID NO 100
<211> LENGTH: 255 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 100 Val
Val Gly Gly Glu Asp Ala Lys Pro Gly Gln Phe Pro Trp Gln Val 1 5 10
15 Val Leu Asn Gly Lys Val Asp Ala Phe Cys Gly Gly Ser Ile Val Asn
20 25 30 Glu Lys Trp Ile Val Thr Ala Ala His Cys Val Glu Glu Thr
Thr Gly 35 40 45 Val Lys Ile Thr Val Val Ala Gly Glu His Asn Ile
Glu Glu Thr Glu 50 55 60 His Thr Glu Gln Lys Arg Asn Val Ile Arg
Ile Ile Pro His His Asn 65 70 75 80 Tyr Asn Asn Asn Ala Ala Ala Ala
Ala Ala Ile Asn Lys Tyr Asn His 85 90 95 Asp Ile Ala Leu Leu Glu
Leu Asp Glu Pro Leu Val Leu Asn Ser Tyr 100 105 110 Val Thr Pro Ile
Cys Ile Ala Asp Lys Glu Tyr Thr Thr Thr Asn Asn 115 120 125 Asn Ile
Ile Ile Phe Leu Lys Phe Gly Ser Gly Tyr Val Ser Gly Trp 130 135 140
Gly Arg Val Phe His Lys Gly Arg Ser Ala Leu Val Leu Gln Tyr Leu 145
150 155 160 Arg Val Pro Leu Val Asp Arg Ala Thr Cys Leu Arg Ser Thr
Lys Phe 165 170 175 Thr Ile Tyr Asn Asn Met Phe Cys Ala Gly Gly Phe
Phe His Glu Gly 180 185 190 Gly Gly Arg Arg Asp Ser Cys Gln Gly Asp
Ser Gly Gly Pro His Val 195 200 205 Thr Glu Val Glu Gly Thr Ser Phe
Leu Thr Gly Ile Ile Ser Trp Gly 210 215 220 Glu Glu Cys Ala Ala Met
Met Lys Gly Lys Tyr Gly Ile Tyr Thr Lys 225 230 235 240 Val Ser Arg
Tyr Val Asn Trp Ile Lys Glu Lys Thr Lys Leu Thr 245 250 255
<210> SEQ ID NO 101 <211> LENGTH: 57 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 101 Met Thr Cys Asn Ile Lys Asn Gly Arg Cys Glu Gln Phe
Cys Lys Asn 1 5 10 15 Ser Ala Asp Asn Lys Val Val Cys Ser Cys Thr
Glu Gly Tyr Arg Leu 20 25 30 Ala Glu Asn Gln Lys Ser Cys Glu Pro
Ala Val Pro Phe Pro Cys Gly 35 40 45 Arg Val Ser Val Ser Gln Thr
Ser Lys 50 55 <210> SEQ ID NO 102 <211> LENGTH: 496
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <400> SEQUENCE: 102 Glu Phe Ala Arg Pro Cys Ile
Pro Lys Ser Phe Gly Tyr Ser Ser Val 1 5 10 15 Val Cys Val Cys Asn
Ala Thr Tyr Cys Asp Ser Phe Asp Pro Pro Ala 20 25 30 Leu Gly Thr
Phe Ser Arg Tyr Glu Ser Thr Arg Ser Gly Arg Arg Met 35 40 45 Glu
Leu Ser Met Gly Pro Ile Gln Ala Asn His Thr Gly Thr Gly Leu 50 55
60 Leu Leu Thr Leu Gln Pro Glu Gln Lys Phe Gln Lys Val Lys Gly Phe
65 70 75 80 Gly Gly Ala Met Thr Asp Ala Ala Ala Leu Asn Ile Leu Ala
Leu Ser 85 90 95 Pro Pro Ala Gln Asn Leu Leu Leu Lys Ser Tyr Phe
Ser Glu Glu Gly 100 105 110 Ile Gly Tyr Asn Ile Ile Arg Val Pro Met
Ala Ser Cys Asp Phe Ser 115 120 125 Ile Arg Thr Tyr Thr Tyr Ala Asp
Thr Pro Asp Asp Phe Gln Leu His 130 135 140 Asn Phe Ser Leu Pro Glu
Glu Asp Thr Lys Leu Lys Ile Pro Leu Ile 145 150 155 160 His Arg Ala
Leu Gln Leu Ala Gln Arg Pro Val Ser Leu Leu Ala Ser 165 170 175 Pro
Trp Thr Ser Pro Thr Trp Leu Lys Thr Asn Gly Ala Val Asn Gly 180 185
190 Lys Gly Ser Leu Lys Gly Gln Pro Gly Asp Ile Tyr His Gln Thr Trp
195 200 205 Ala Arg Tyr Phe Val Lys Phe Leu Asp Ala Tyr Ala Glu His
Lys Leu 210 215 220 Gln Phe Trp Ala Val Thr Ala Glu Asn Glu Pro Ser
Ala Gly Leu Leu
225 230 235 240 Ser Gly Tyr Pro Phe Gln Cys Leu Gly Phe Thr Pro Glu
His Gln Arg 245 250 255 Asp Phe Ile Ala Arg Asp Leu Gly Pro Thr Leu
Ala Asn Ser Thr His 260 265 270 His Asn Val Arg Leu Leu Met Leu Asp
Asp Gln Arg Leu Leu Leu Pro 275 280 285 His Trp Ala Lys Val Val Leu
Thr Asp Pro Glu Ala Ala Lys Tyr Val 290 295 300 His Gly Ile Ala Val
His Trp Tyr Leu Asp Phe Leu Ala Pro Ala Lys 305 310 315 320 Ala Thr
Leu Gly Glu Thr His Arg Leu Phe Pro Asn Thr Met Leu Phe 325 330 335
Ala Ser Glu Ala Cys Val Gly Ser Lys Phe Trp Glu Gln Ser Val Arg 340
345 350 Leu Gly Ser Trp Asp Arg Gly Met Gln Tyr Ser His Ser Ile Ile
Thr 355 360 365 Asn Leu Leu Tyr His Val Val Gly Trp Thr Asp Trp Asn
Leu Ala Leu 370 375 380 Asn Pro Glu Gly Gly Pro Asn Trp Val Arg Asn
Phe Val Asp Ser Pro 385 390 395 400 Ile Ile Val Asp Ile Thr Lys Asp
Thr Phe Tyr Lys Gln Pro Met Phe 405 410 415 Tyr His Leu Gly His Phe
Ser Lys Phe Ile Pro Glu Gly Ser Gln Arg 420 425 430 Val Gly Leu Val
Ala Ser Gln Lys Asn Asp Leu Asp Ala Val Ala Leu 435 440 445 Met His
Pro Asp Gly Ser Ala Val Val Val Val Leu Asn Arg Ser Ser 450 455 460
Lys Asp Val Pro Leu Thr Ile Lys Asp Pro Ala Val Gly Phe Leu Glu 465
470 475 480 Thr Ile Ser Pro Gly Tyr Ser Ile His Thr Tyr Leu Trp His
Arg Gln 485 490 495 <210> SEQ ID NO 103 <211> LENGTH:
390 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <400> SEQUENCE: 103 Leu Asp Asn Gly Leu Ala Arg
Thr Pro Thr Met Gly Trp Leu His Trp 1 5 10 15 Glu Arg Phe Met Cys
Asn Leu Asp Cys Gln Glu Glu Pro Asp Ser Cys 20 25 30 Ile Ser Glu
Lys Leu Phe Met Glu Met Ala Glu Leu Met Val Ser Glu 35 40 45 Gly
Trp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys Ile Asp Asp Cys Trp 50 55
60 Met Ala Pro Gln Arg Asp Ser Glu Gly Arg Leu Gln Ala Asp Pro Gln
65 70 75 80 Arg Phe Pro His Gly Ile Arg Gln Leu Ala Asn Tyr Val His
Ser Lys 85 90 95 Gly Leu Lys Leu Gly Ile Tyr Ala Asp Val Gly Asn
Lys Thr Cys Ala 100 105 110 Gly Phe Pro Gly Ser Phe Gly Tyr Tyr Asp
Ile Asp Ala Gln Thr Phe 115 120 125 Ala Asp Trp Gly Val Asp Leu Leu
Lys Phe Asp Gly Cys Tyr Cys Asp 130 135 140 Ser Leu Glu Asn Leu Ala
Asp Gly Tyr Lys His Met Ser Leu Ala Leu 145 150 155 160 Asn Arg Thr
Gly Arg Ser Ile Val Tyr Ser Cys Glu Trp Pro Leu Tyr 165 170 175 Met
Trp Pro Phe Gln Lys Pro Asn Tyr Thr Glu Ile Arg Gln Tyr Cys 180 185
190 Asn His Trp Arg Asn Phe Ala Asp Ile Asp Asp Ser Trp Lys Ser Ile
195 200 205 Lys Ser Ile Leu Asp Trp Thr Ser Phe Asn Gln Glu Arg Ile
Val Asp 210 215 220 Val Ala Gly Pro Gly Gly Trp Asn Asp Pro Asp Met
Leu Val Ile Gly 225 230 235 240 Asn Phe Gly Leu Ser Trp Asn Gln Gln
Val Thr Gln Met Ala Leu Trp 245 250 255 Ala Ile Met Ala Ala Pro Leu
Phe Met Ser Asn Asp Leu Arg His Ile 260 265 270 Ser Pro Gln Ala Lys
Ala Leu Leu Gln Asp Lys Asp Val Ile Ala Ile 275 280 285 Asn Gln Asp
Pro Leu Gly Lys Gln Gly Tyr Gln Leu Arg Gln Gly Asp 290 295 300 Asn
Phe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu Ala Trp Ala Val 305 310
315 320 Ala Met Ile Asn Arg Gln Glu Ile Gly Gly Pro Arg Ser Tyr Thr
Ile 325 330 335 Ala Val Ala Ser Leu Gly Lys Gly Val Ala Cys Asn Pro
Ala Cys Phe 340 345 350 Ile Thr Gln Leu Leu Pro Val Lys Arg Lys Leu
Gly Phe Tyr Glu Trp 355 360 365 Thr Ser Arg Leu Arg Ser His Ile Asn
Pro Thr Gly Thr Val Leu Leu 370 375 380 Gln Leu Glu Asn Thr Met 385
390 <210> SEQ ID NO 104 <211> LENGTH: 479 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide
<400> SEQUENCE: 104 Arg Pro Pro Asn Ile Val Leu Ile Phe Ala
Asp Asp Leu Gly Tyr Gly 1 5 10 15 Asp Leu Gly Cys Tyr Gly His Pro
Ser Ser Thr Thr Pro Asn Leu Asp 20 25 30 Gln Leu Ala Ala Gly Gly
Leu Arg Phe Thr Asp Phe Tyr Val Pro Val 35 40 45 Ser Leu Pro Ser
Arg Ala Ala Leu Leu Thr Gly Arg Leu Pro Val Arg 50 55 60 Met Gly
Met Tyr Pro Gly Val Leu Val Pro Ser Ser Arg Gly Gly Leu 65 70 75 80
Pro Leu Glu Glu Val Thr Val Ala Glu Val Leu Ala Ala Arg Gly Tyr 85
90 95 Leu Thr Gly Met Ala Gly Lys Trp His Leu Gly Val Gly Pro Glu
Gly 100 105 110 Ala Phe Leu Pro Pro His Gln Gly Phe His Arg Phe Leu
Gly Ile Pro 115 120 125 Tyr Ser His Asp Gln Gly Pro Cys Gln Asn Leu
Thr Cys Phe Pro Pro 130 135 140 Ala Thr Pro Cys Asp Gly Gly Cys Asp
Gln Gly Leu Val Pro Ile Pro 145 150 155 160 Leu Leu Ala Asn Leu Ser
Val Glu Ala Gln Pro Pro Trp Leu Pro Gly 165 170 175 Leu Glu Ala Arg
Tyr Met Ala Phe Ala His Asp Leu Met Ala Asp Ala 180 185 190 Gln Arg
Gln Asp Arg Pro Phe Phe Leu Tyr Tyr Ala Ser His His Thr 195 200 205
His Tyr Pro Gln Phe Ser Gly Gln Ser Phe Ala Glu Arg Ser Gly Arg 210
215 220 Gly Pro Phe Gly Asp Ser Leu Met Glu Leu Asp Ala Ala Val Gly
Thr 225 230 235 240 Leu Met Thr Ala Ile Gly Asp Leu Gly Leu Leu Glu
Glu Thr Leu Val 245 250 255 Ile Phe Thr Ala Asp Asn Gly Pro Glu Thr
Met Arg Met Ser Arg Gly 260 265 270 Gly Cys Ser Gly Leu Leu Arg Cys
Gly Lys Gly Thr Thr Tyr Glu Gly 275 280 285 Gly Val Arg Glu Pro Ala
Leu Ala Phe Trp Pro Gly His Ile Ala Pro 290 295 300 Gly Val Thr His
Glu Leu Ala Ser Ser Leu Asp Leu Leu Pro Thr Leu 305 310 315 320 Ala
Ala Leu Ala Gly Ala Pro Leu Pro Asn Val Thr Leu Asp Gly Phe 325 330
335 Asp Leu Ser Pro Leu Leu Leu Gly Thr Gly Lys Ser Pro Arg Gln Ser
340 345 350 Leu Phe Phe Tyr Pro Ser Tyr Pro Asp Glu Val Arg Gly Val
Phe Ala 355 360 365 Val Arg Thr Gly Lys Tyr Lys Ala His Phe Phe Thr
Gln Gly Ser Ala 370 375 380 His Ser Asp Thr Thr Ala Asp Pro Ala Cys
His Ala Ser Ser Ser Leu 385 390 395 400 Thr Ala His Glu Pro Pro Leu
Leu Tyr Asp Leu Ser Lys Asp Pro Gly 405 410 415 Glu Asn Tyr Asn Leu
Leu Gly Ala Thr Pro Glu Val Leu Gln Ala Leu 420 425 430 Lys Gln Leu
Gln Leu Leu Lys Ala Gln Leu Asp Ala Ala Val Thr Phe 435 440 445 Gly
Pro Ser Gln Val Ala Arg Gly Glu Asp Pro Ala Leu Gln Ile Cys 450 455
460 Cys His Pro Gly Cys Thr Pro Arg Pro Ala Cys Cys His Cys Pro 465
470 475 <210> SEQ ID NO 105 <211> LENGTH: 474
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <400> SEQUENCE: 105 Ser Arg Pro Pro His Leu Val
Phe Leu Leu Ala Asp Asp Leu Gly Trp 1 5 10 15 Asn Asp Val Gly Phe
His Gly Ser Arg Ile Arg Thr Pro His Leu Asp 20 25 30
Ala Leu Ala Ala Gly Gly Val Leu Leu Asp Asn Tyr Tyr Thr Gln Pro 35
40 45 Leu Thr Pro Ser Arg Ser Gln Leu Leu Thr Gly Arg Tyr Gln Ile
Arg 50 55 60 Thr Gly Leu Gln His Gln Ile Ile Trp Pro Cys Gln Pro
Ser Cys Val 65 70 75 80 Pro Leu Asp Glu Lys Leu Leu Pro Gln Leu Leu
Lys Glu Ala Gly Tyr 85 90 95 Thr Thr His Met Val Gly Lys Trp His
Leu Gly Met Tyr Arg Lys Glu 100 105 110 Cys Leu Pro Thr Arg Arg Gly
Phe Asp Thr Tyr Phe Gly Tyr Leu Leu 115 120 125 Gly Ser Glu Asp Tyr
Tyr Ser His Glu Arg Cys Thr Leu Ile Asp Ala 130 135 140 Leu Asn Val
Thr Arg Cys Ala Leu Asp Phe Arg Asp Gly Glu Glu Val 145 150 155 160
Ala Thr Gly Tyr Lys Asn Met Tyr Ser Thr Asn Ile Phe Thr Lys Arg 165
170 175 Ala Ile Ala Leu Ile Thr Asn His Pro Pro Glu Lys Pro Leu Phe
Leu 180 185 190 Tyr Leu Ala Leu Gln Ser Val His Glu Pro Leu Gln Val
Pro Glu Glu 195 200 205 Tyr Leu Lys Pro Tyr Asp Phe Ile Gln Asp Lys
Asn Arg His His Tyr 210 215 220 Ala Gly Met Val Ser Leu Met Asp Glu
Ala Val Gly Asn Val Thr Ala 225 230 235 240 Ala Leu Lys Ser Ser Gly
Leu Trp Asn Asn Thr Val Phe Ile Phe Ser 245 250 255 Thr Asp Asn Gly
Gly Gln Thr Leu Ala Gly Gly Asn Asn Trp Pro Leu 260 265 270 Arg Gly
Arg Lys Trp Ser Leu Trp Glu Gly Gly Val Arg Gly Val Gly 275 280 285
Phe Val Ala Ser Pro Leu Leu Lys Gln Lys Gly Val Lys Asn Arg Glu 290
295 300 Leu Ile His Ile Ser Asp Trp Leu Pro Thr Leu Val Lys Leu Ala
Arg 305 310 315 320 Gly His Thr Asn Gly Thr Lys Pro Leu Asp Gly Phe
Asp Val Trp Lys 325 330 335 Thr Ile Ser Glu Gly Ser Pro Ser Pro Arg
Ile Glu Leu Leu His Asn 340 345 350 Ile Asp Pro Asn Phe Val Asp Ser
Ser Pro Cys Ser Ala Phe Asn Thr 355 360 365 Ser Val His Ala Ala Ile
Arg His Gly Asn Trp Lys Leu Leu Thr Gly 370 375 380 Tyr Pro Gly Cys
Gly Tyr Trp Phe Pro Pro Pro Ser Gln Tyr Asn Val 385 390 395 400 Ser
Glu Ile Pro Ser Ser Asp Pro Pro Thr Lys Thr Leu Trp Leu Phe 405 410
415 Asp Ile Asp Arg Asp Pro Glu Glu Arg His Asp Leu Ser Arg Glu Tyr
420 425 430 Pro His Ile Val Thr Lys Leu Leu Ser Arg Leu Gln Phe Tyr
His Lys 435 440 445 His Ser Val Pro Val Tyr Phe Pro Ala Gln Asp Pro
Arg Cys Asp Pro 450 455 460 Lys Ala Thr Gly Val Trp Gly Pro Trp Met
465 470 <210> SEQ ID NO 106 <211> LENGTH: 492
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <400> SEQUENCE: 106 Leu Trp Pro Trp Pro Gln Asn
Phe Gln Thr Ser Asp Gln Arg Tyr Val 1 5 10 15 Leu Tyr Pro Asn Asn
Phe Gln Phe Gln Tyr Asp Val Ser Ser Ala Ala 20 25 30 Gln Pro Gly
Cys Ser Val Leu Asp Glu Ala Phe Gln Arg Tyr Arg Asp 35 40 45 Leu
Leu Phe Gly Thr Leu Glu Lys Asn Val Leu Val Val Ser Val Val 50 55
60 Thr Pro Gly Cys Asn Gln Leu Pro Thr Leu Glu Ser Val Glu Asn Tyr
65 70 75 80 Thr Leu Thr Ile Asn Asp Asp Gln Cys Leu Leu Leu Ser Glu
Thr Val 85 90 95 Trp Gly Ala Leu Arg Gly Leu Glu Thr Phe Ser Gln
Leu Val Trp Lys 100 105 110 Ser Ala Glu Gly Thr Phe Phe Ile Asn Lys
Thr Glu Ile Glu Asp Phe 115 120 125 Pro Arg Phe Pro His Arg Gly Leu
Leu Leu Asp Thr Ser Arg His Tyr 130 135 140 Leu Pro Leu Ser Ser Ile
Leu Asp Thr Leu Asp Val Met Ala Tyr Asn 145 150 155 160 Lys Leu Asn
Val Phe His Trp His Leu Val Asp Asp Pro Ser Phe Pro 165 170 175 Tyr
Glu Ser Phe Thr Phe Pro Glu Leu Met Arg Lys Gly Ser Tyr Asn 180 185
190 Pro Val Thr His Ile Tyr Thr Ala Gln Asp Val Lys Glu Val Ile Glu
195 200 205 Tyr Ala Arg Leu Arg Gly Ile Arg Val Leu Ala Glu Phe Asp
Thr Pro 210 215 220 Gly His Thr Leu Ser Trp Gly Pro Gly Ile Pro Gly
Leu Leu Thr Pro 225 230 235 240 Cys Tyr Ser Gly Ser Glu Pro Ser Gly
Thr Phe Gly Pro Val Asn Pro 245 250 255 Ser Leu Asn Asn Thr Tyr Glu
Phe Met Ser Thr Phe Phe Leu Glu Val 260 265 270 Ser Ser Val Phe Pro
Asp Phe Tyr Leu His Leu Gly Gly Asp Glu Val 275 280 285 Asp Phe Thr
Cys Trp Lys Ser Asn Pro Glu Ile Gln Asp Phe Met Arg 290 295 300 Lys
Lys Gly Phe Gly Glu Asp Phe Lys Gln Leu Glu Ser Phe Tyr Ile 305 310
315 320 Gln Thr Leu Leu Asp Ile Val Ser Ser Tyr Gly Lys Gly Tyr Val
Val 325 330 335 Trp Gln Glu Val Phe Asp Asn Lys Val Lys Ile Gln Pro
Asp Thr Ile 340 345 350 Ile Gln Val Trp Arg Glu Asp Ile Pro Val Asn
Tyr Met Lys Glu Leu 355 360 365 Glu Leu Val Thr Lys Ala Gly Phe Arg
Ala Leu Leu Ser Ala Pro Trp 370 375 380 Tyr Leu Asn Arg Ile Ser Tyr
Gly Pro Asp Trp Lys Asp Phe Tyr Val 385 390 395 400 Val Glu Pro Leu
Ala Phe Glu Gly Thr Pro Glu Gln Lys Ala Leu Val 405 410 415 Ile Gly
Gly Glu Ala Cys Met Trp Gly Glu Tyr Val Asp Asn Thr Asn 420 425 430
Leu Val Pro Arg Leu Trp Pro Arg Ala Gly Ala Val Ala Glu Arg Leu 435
440 445 Trp Ser Asn Lys Leu Thr Ser Asp Leu Thr Phe Ala Tyr Glu Arg
Leu 450 455 460 Ser His Phe Arg Cys Glu Leu Leu Arg Arg Gly Val Gln
Ala Gln Pro 465 470 475 480 Leu Asn Val Gly Phe Cys Glu Gln Glu Phe
Glu Gln 485 490 <210> SEQ ID NO 107 <211> LENGTH: 492
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <400> SEQUENCE: 107 Leu Trp Pro Trp Pro Gln Asn
Phe Gln Thr Ser Asp Gln Arg Tyr Val 1 5 10 15 Leu Tyr Pro Asn Asn
Phe Gln Phe Gln Tyr Asp Val Ser Ser Ala Ala 20 25 30 Gln Pro Gly
Cys Ser Val Leu Asp Glu Ala Phe Gln Arg Tyr Arg Asp 35 40 45 Leu
Leu Phe Gly Thr Leu Glu Lys Asn Val Leu Val Val Ser Val Val 50 55
60 Thr Pro Gly Cys Asn Gln Leu Pro Thr Leu Glu Ser Val Glu Asn Tyr
65 70 75 80 Thr Leu Thr Ile Asn Asp Asp Gln Cys Leu Leu Leu Ser Glu
Thr Val 85 90 95 Trp Gly Ala Leu Arg Gly Leu Glu Thr Phe Ser Gln
Leu Val Trp Lys 100 105 110 Ser Ala Glu Gly Thr Phe Phe Ile Asn Lys
Thr Glu Ile Glu Asp Phe 115 120 125 Pro Arg Phe Pro His Arg Gly Leu
Leu Leu Asp Thr Ser Arg His Tyr 130 135 140 Leu Pro Leu Ser Ser Ile
Leu Asp Thr Leu Asp Val Met Ala Tyr Asn 145 150 155 160 Lys Leu Asn
Val Phe His Trp His Leu Val Asp Asp Pro Ser Phe Pro 165 170 175 Tyr
Glu Ser Phe Thr Phe Pro Glu Leu Met Arg Lys Gly Ser Tyr Asn 180 185
190 Pro Val Thr His Ile Tyr Thr Ala Gln Asp Val Lys Glu Val Ile Glu
195 200 205 Tyr Ala Arg Leu Arg Gly Ile Arg Val Leu Ala Glu Phe Asp
Thr Pro 210 215 220 Gly His Thr Leu Ser Trp Gly Pro Gly Ile Pro Gly
Leu Leu Thr Pro 225 230 235 240 Cys Tyr Ser Gly Ser Glu Pro Ser Gly
Thr Phe Gly Pro Val Asn Pro 245 250 255 Ser Leu Asn Asn Thr Tyr Glu
Phe Met Ser Thr Phe Phe Leu Glu Val 260 265 270 Ser Ser Val Phe Pro
Asp Phe Tyr Leu His Leu Gly Gly Asp Glu Val 275 280 285
Asp Phe Thr Cys Trp Lys Ser Asn Pro Glu Ile Gln Asp Phe Met Arg 290
295 300 Lys Lys Gly Phe Gly Glu Asp Phe Lys Gln Leu Glu Ser Phe Tyr
Ile 305 310 315 320 Gln Thr Leu Leu Asp Ile Val Ser Ser Tyr Gly Lys
Gly Tyr Val Val 325 330 335 Trp Gln Glu Val Phe Asp Asn Lys Val Lys
Ile Gln Pro Asp Thr Ile 340 345 350 Ile Gln Val Trp Arg Glu Asp Ile
Pro Val Asn Tyr Met Lys Glu Leu 355 360 365 Glu Leu Val Thr Lys Ala
Gly Phe Arg Ala Leu Leu Ser Ala Pro Trp 370 375 380 Tyr Leu Asn Arg
Ile Ser Tyr Gly Pro Asp Trp Lys Asp Phe Tyr Val 385 390 395 400 Val
Glu Pro Leu Ala Phe Glu Gly Thr Pro Glu Gln Lys Ala Leu Val 405 410
415 Ile Gly Gly Glu Ala Cys Met Trp Gly Glu Tyr Val Asp Asn Thr Asn
420 425 430 Leu Val Pro Arg Leu Trp Pro Arg Ala Gly Ala Val Ala Glu
Arg Leu 435 440 445 Trp Ser Asn Lys Leu Thr Ser Asp Leu Thr Phe Ala
Tyr Glu Arg Leu 450 455 460 Ser His Phe Arg Cys Glu Leu Leu Arg Arg
Gly Val Gln Ala Gln Pro 465 470 475 480 Leu Asn Val Gly Phe Cys Glu
Gln Glu Phe Glu Gln 485 490 <210> SEQ ID NO 108 <211>
LENGTH: 480 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Polypeptide <400> SEQUENCE: 108 Pro Ala Leu Trp Pro
Leu Pro Leu Ser Val Lys Met Thr Pro Asn Leu 1 5 10 15 Leu His Leu
Ala Pro Glu Asn Phe Tyr Ile Ser His Ser Pro Asn Ser 20 25 30 Thr
Ala Gly Pro Ser Cys Thr Leu Leu Glu Glu Ala Phe Arg Arg Tyr 35 40
45 His Gly Tyr Ile Phe Gly Thr Gln Val Gln Gln Leu Leu Val Ser Ile
50 55 60 Thr Leu Gln Ser Glu Cys Asp Ala Phe Pro Asn Ile Ser Ser
Asp Glu 65 70 75 80 Ser Tyr Thr Leu Leu Val Lys Glu Pro Val Ala Val
Leu Lys Ala Asn 85 90 95 Arg Val Trp Gly Ala Leu Arg Gly Leu Glu
Thr Phe Ser Gln Leu Val 100 105 110 Tyr Gln Asp Ser Tyr Gly Thr Phe
Thr Ile Asn Glu Ser Thr Ile Ile 115 120 125 Asp Ser Pro Arg Phe Ser
His Arg Gly Ile Leu Ile Asp Thr Ser Arg 130 135 140 His Tyr Leu Pro
Val Lys Ile Ile Leu Lys Thr Leu Asp Ala Met Ala 145 150 155 160 Phe
Asn Lys Phe Asn Val Leu His Trp His Ile Val Asp Asp Gln Ser 165 170
175 Phe Pro Tyr Gln Ser Ile Thr Phe Pro Glu Leu Ser Asn Lys Gly Ser
180 185 190 Tyr Ser Leu Ser His Val Tyr Thr Pro Asn Asp Val Arg Met
Val Ile 195 200 205 Glu Tyr Ala Arg Leu Arg Gly Ile Arg Val Leu Pro
Glu Phe Asp Thr 210 215 220 Pro Gly His Thr Leu Ser Trp Gly Lys Gly
Gln Lys Asp Leu Leu Thr 225 230 235 240 Pro Cys Tyr Ser Asp Ser Phe
Gly Pro Ile Asn Pro Thr Leu Asn Thr 245 250 255 Thr Tyr Ser Phe Leu
Thr Thr Phe Phe Lys Glu Ile Ser Glu Val Phe 260 265 270 Pro Asp Gln
Phe Ile His Leu Gly Gly Asp Glu Val Glu Phe Lys Cys 275 280 285 Trp
Glu Ser Asn Pro Lys Ile Gln Asp Phe Met Arg Gln Lys Gly Phe 290 295
300 Gly Thr Asp Phe Lys Lys Leu Glu Ser Phe Tyr Ile Gln Lys Val Leu
305 310 315 320 Asp Ile Ile Ala Thr Ile Asn Lys Gly Ser Ile Val Trp
Gln Glu Val 325 330 335 Phe Asp Asp Lys Ala Lys Leu Ala Pro Gly Thr
Ile Val Glu Val Trp 340 345 350 Lys Asp Ser Ala Tyr Pro Glu Glu Leu
Ser Arg Val Thr Ala Ser Gly 355 360 365 Phe Pro Val Ile Leu Ser Ala
Pro Trp Tyr Leu Asp Leu Ile Ser Tyr 370 375 380 Gly Gln Asp Trp Arg
Lys Tyr Tyr Lys Val Glu Pro Leu Asp Phe Gly 385 390 395 400 Gly Thr
Gln Lys Gln Lys Gln Leu Phe Ile Gly Gly Glu Ala Cys Leu 405 410 415
Trp Gly Glu Tyr Val Asp Ala Thr Asn Leu Thr Pro Arg Leu Trp Pro 420
425 430 Arg Ala Ser Ala Val Gly Glu Arg Leu Trp Ser Ser Lys Asp Val
Arg 435 440 445 Asp Met Asp Asp Ala Tyr Asp Arg Leu Thr Arg His Arg
Cys Arg Met 450 455 460 Val Glu Arg Gly Ile Ala Ala Gln Pro Leu Tyr
Ala Gly Tyr Cys Asn 465 470 475 480 <210> SEQ ID NO 109
<211> LENGTH: 481 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 109 Pro
Ala Leu Trp Pro Leu Pro Leu Ser Val Lys Met Thr Pro Asn Leu 1 5 10
15 Leu His Leu Ala Pro Glu Asn Phe Tyr Ile Ser His Ser Pro Asn Ser
20 25 30 Thr Ala Gly Pro Ser Cys Thr Leu Leu Glu Glu Ala Phe Arg
Arg Tyr 35 40 45 His Gly Tyr Ile Phe Gly Thr Gln Val Gln Gln Leu
Leu Val Ser Ile 50 55 60 Thr Leu Gln Ser Glu Cys Asp Ala Phe Pro
Asn Ile Ser Ser Asp Glu 65 70 75 80 Ser Tyr Thr Leu Leu Val Lys Glu
Pro Val Ala Val Leu Lys Ala Asn 85 90 95 Arg Val Trp Gly Ala Leu
Arg Gly Leu Glu Thr Phe Ser Gln Leu Val 100 105 110 Tyr Gln Asp Ser
Tyr Gly Thr Phe Thr Ile Asn Glu Ser Thr Ile Ile 115 120 125 Asp Ser
Pro Arg Phe Ser His Arg Gly Ile Leu Ile Asp Thr Ser Arg 130 135 140
His Tyr Leu Pro Val Lys Ile Ile Leu Lys Thr Leu Asp Ala Met Ala 145
150 155 160 Phe Asn Lys Phe Asn Val Leu His Trp His Ile Val Asp Asp
Gln Ser 165 170 175 Phe Pro Tyr Gln Ser Ile Thr Phe Pro Glu Leu Ser
Asn Lys Gly Ser 180 185 190 Tyr Ser Leu Ser His Val Tyr Thr Pro Asn
Asp Val Arg Met Val Ile 195 200 205 Glu Tyr Ala Arg Leu Arg Gly Ile
Arg Val Leu Pro Glu Phe Asp Thr 210 215 220 Pro Gly His Thr Leu Ser
Trp Gly Lys Gly Gln Lys Asp Leu Leu Thr 225 230 235 240 Pro Cys Tyr
Ser Leu Asp Ser Phe Gly Pro Ile Asn Pro Thr Leu Asn 245 250 255 Thr
Thr Tyr Ser Phe Leu Thr Thr Phe Phe Lys Glu Ile Ser Glu Val 260 265
270 Phe Pro Asp Gln Phe Ile His Leu Gly Gly Asp Glu Val Glu Phe Lys
275 280 285 Cys Trp Glu Ser Asn Pro Lys Ile Gln Asp Phe Met Arg Gln
Lys Gly 290 295 300 Phe Gly Thr Asp Phe Lys Lys Leu Glu Ser Phe Tyr
Ile Gln Lys Val 305 310 315 320 Leu Asp Ile Ile Ala Thr Ile Asn Lys
Gly Ser Ile Val Trp Gln Glu 325 330 335 Val Phe Asp Asp Lys Ala Lys
Leu Ala Pro Gly Thr Ile Val Glu Val 340 345 350 Trp Lys Asp Ser Ala
Tyr Pro Glu Glu Leu Ser Arg Val Thr Ala Ser 355 360 365 Gly Phe Pro
Val Ile Leu Ser Ala Pro Trp Tyr Leu Asp Leu Ile Ser 370 375 380 Tyr
Gly Gln Asp Trp Arg Lys Tyr Tyr Lys Val Glu Pro Leu Asp Phe 385 390
395 400 Gly Gly Thr Gln Lys Gln Lys Gln Leu Phe Ile Gly Gly Glu Ala
Cys 405 410 415 Leu Trp Gly Glu Tyr Val Asp Ala Thr Asn Leu Thr Pro
Arg Leu Trp 420 425 430 Pro Arg Ala Ser Ala Val Gly Glu Arg Leu Trp
Ser Ser Lys Asp Val 435 440 445 Arg Asp Met Asp Asp Ala Tyr Asp Arg
Leu Thr Arg His Arg Cys Arg 450 455 460 Met Val Glu Arg Gly Ile Ala
Ala Gln Pro Leu Tyr Ala Gly Tyr Cys 465 470 475 480 Asn <210>
SEQ ID NO 110 <211> LENGTH: 492 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 110
Leu Trp Pro Trp Pro Gln Asn Phe Gln Thr Ser Asp Gln Arg Tyr Val 1 5
10 15 Leu Tyr Pro Asn Asn Phe Gln Phe Gln Tyr Asp Val Ser Ser Ala
Ala 20 25 30 Gln Pro Gly Cys Ser Val Leu Asp Glu Ala Phe Gln Arg
Tyr Arg Asp 35 40 45 Leu Leu Phe Gly Thr Leu Glu Lys Asn Val Leu
Val Val Ser Val Val 50 55 60 Thr Pro Gly Cys Asn Gln Leu Pro Thr
Leu Glu Ser Val Glu Asn Tyr 65 70 75 80 Thr Leu Thr Ile Asn Asp Asp
Gln Cys Leu Leu Leu Ser Glu Thr Val 85 90 95 Trp Gly Ala Leu Arg
Gly Leu Glu Thr Phe Ser Gln Leu Val Trp Lys 100 105 110 Ser Ala Glu
Gly Thr Phe Phe Ile Asn Lys Thr Glu Ile Glu Asp Phe 115 120 125 Pro
Arg Phe Pro His Arg Gly Leu Leu Leu Asp Thr Ser Arg His Tyr 130 135
140 Leu Pro Leu Ser Ser Ile Leu Asp Thr Leu Asp Val Met Ala Tyr Asn
145 150 155 160 Lys Leu Asn Val Phe His Trp His Leu Val Asp Asp Pro
Ser Phe Pro 165 170 175 Tyr Glu Ser Phe Thr Phe Pro Glu Leu Met Arg
Lys Gly Ser Tyr Asn 180 185 190 Pro Val Thr His Ile Tyr Thr Ala Gln
Asp Val Lys Glu Val Ile Glu 195 200 205 Tyr Ala Arg Leu Arg Gly Ile
Arg Val Leu Ala Glu Phe Asp Thr Pro 210 215 220 Gly His Thr Leu Ser
Trp Gly Pro Gly Ile Pro Gly Leu Leu Thr Pro 225 230 235 240 Cys Tyr
Ser Gly Ser Glu Pro Ser Gly Thr Phe Gly Pro Val Asn Pro 245 250 255
Ser Leu Asn Asn Thr Tyr Glu Phe Met Ser Thr Phe Phe Leu Glu Val 260
265 270 Ser Ser Val Phe Pro Asp Phe Tyr Leu His Leu Gly Gly Asp Glu
Val 275 280 285 Asp Phe Thr Cys Trp Lys Ser Asn Pro Glu Ile Gln Asp
Phe Met Arg 290 295 300 Lys Lys Gly Phe Gly Glu Asp Phe Lys Gln Leu
Glu Ser Phe Tyr Ile 305 310 315 320 Gln Thr Leu Leu Asp Ile Val Ser
Ser Tyr Gly Lys Gly Tyr Val Val 325 330 335 Trp Gln Glu Val Phe Asp
Asn Lys Val Lys Ile Gln Pro Asp Thr Ile 340 345 350 Ile Gln Val Trp
Arg Glu Asp Ile Pro Val Asn Tyr Met Lys Glu Leu 355 360 365 Glu Leu
Val Thr Lys Ala Gly Phe Arg Ala Leu Leu Ser Ala Pro Trp 370 375 380
Tyr Leu Asn Arg Ile Ser Tyr Gly Pro Asp Trp Lys Asp Phe Tyr Val 385
390 395 400 Val Glu Pro Leu Ala Phe Glu Gly Thr Pro Glu Gln Lys Ala
Leu Val 405 410 415 Ile Gly Gly Glu Ala Cys Met Trp Gly Glu Tyr Val
Asp Asn Thr Asn 420 425 430 Leu Val Pro Arg Leu Trp Pro Arg Ala Gly
Ala Val Ala Glu Arg Leu 435 440 445 Trp Ser Asn Lys Leu Thr Ser Asp
Leu Thr Phe Ala Tyr Glu Arg Leu 450 455 460 Ser His Phe Arg Cys Glu
Leu Leu Arg Arg Gly Val Gln Ala Gln Pro 465 470 475 480 Leu Asn Val
Gly Phe Cys Glu Gln Glu Phe Glu Gln 485 490 <210> SEQ ID NO
111 <211> LENGTH: 307 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 111
Val Pro Trp Phe Pro Arg Thr Ile Gln Glu Leu Asp Arg Phe Ala Asn 1 5
10 15 Gln Ile Leu Ser Tyr Gly Ala Glu Leu Asp Ala Asp His Pro Gly
Phe 20 25 30 Lys Asp Pro Val Tyr Arg Ala Arg Arg Lys Gln Phe Ala
Asp Ile Ala 35 40 45 Tyr Asn Tyr Arg His Gly Gln Pro Ile Pro Arg
Val Glu Tyr Met Glu 50 55 60 Glu Glu Lys Lys Thr Trp Gly Thr Val
Phe Lys Thr Leu Lys Ser Leu 65 70 75 80 Tyr Lys Thr His Ala Cys Tyr
Glu Tyr Asn His Ile Phe Pro Leu Leu 85 90 95 Glu Lys Tyr Cys Gly
Phe His Glu Asp Asn Ile Pro Gln Leu Glu Asp 100 105 110 Val Ser Gln
Phe Leu Gln Thr Cys Thr Gly Phe Arg Leu Arg Pro Val 115 120 125 Ala
Gly Leu Leu Ser Ser Arg Asp Phe Leu Gly Gly Leu Ala Phe Arg 130 135
140 Val Phe His Cys Thr Gln Tyr Ile Arg His Gly Ser Lys Pro Met Tyr
145 150 155 160 Thr Pro Glu Pro Asp Ile Cys His Glu Leu Leu Gly His
Val Pro Leu 165 170 175 Phe Ser Asp Arg Ser Phe Ala Gln Phe Ser Gln
Glu Ile Gly Leu Ala 180 185 190 Ser Leu Gly Ala Pro Asp Glu Tyr Ile
Glu Lys Leu Ala Thr Ile Tyr 195 200 205 Trp Phe Thr Val Glu Phe Gly
Leu Cys Lys Gln Gly Asp Ser Ile Lys 210 215 220 Ala Tyr Gly Ala Gly
Leu Leu Ser Ser Phe Gly Glu Leu Gln Tyr Cys 225 230 235 240 Leu Ser
Glu Lys Pro Lys Leu Leu Pro Leu Glu Leu Glu Lys Thr Ala 245 250 255
Ile Gln Asn Tyr Thr Val Thr Glu Phe Gln Pro Leu Tyr Tyr Val Ala 260
265 270 Glu Ser Phe Asn Asp Ala Lys Glu Lys Val Arg Asn Phe Ala Ala
Thr 275 280 285 Ile Pro Arg Pro Phe Ser Val Arg Tyr Asp Pro Tyr Thr
Gln Arg Ile 290 295 300 Glu Val Leu 305 <210> SEQ ID NO 112
<211> LENGTH: 452 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 112 Ala
Pro Asp Gln Asp Glu Ile Gln Arg Leu Pro Gly Leu Ala Lys Gln 1 5 10
15 Pro Ser Phe Arg Gln Tyr Ser Gly Tyr Leu Lys Ser Ser Gly Ser Lys
20 25 30 His Leu His Tyr Trp Phe Val Glu Ser Gln Lys Asp Pro Glu
Asn Ser 35 40 45 Pro Val Val Leu Trp Leu Asn Gly Gly Pro Gly Cys
Ser Ser Leu Asp 50 55 60 Gly Leu Leu Thr Glu His Gly Pro Phe Leu
Val Gln Pro Asp Gly Val 65 70 75 80 Thr Leu Glu Tyr Asn Pro Tyr Ser
Trp Asn Leu Ile Ala Asn Val Leu 85 90 95 Tyr Leu Glu Ser Pro Ala
Gly Val Gly Phe Ser Tyr Ser Asp Asp Lys 100 105 110 Phe Tyr Ala Thr
Asn Asp Thr Glu Val Ala Gln Ser Asn Phe Glu Ala 115 120 125 Leu Gln
Asp Phe Phe Arg Leu Phe Pro Glu Tyr Lys Asn Asn Lys Leu 130 135 140
Phe Leu Thr Gly Glu Ser Tyr Ala Gly Ile Tyr Ile Pro Thr Leu Ala 145
150 155 160 Val Leu Val Met Gln Asp Pro Ser Met Asn Leu Gln Gly Leu
Ala Val 165 170 175 Gly Asn Gly Leu Ser Ser Tyr Glu Gln Asn Asp Asn
Ser Leu Val Tyr 180 185 190 Phe Ala Tyr Tyr His Gly Leu Leu Gly Asn
Arg Leu Trp Ser Ser Leu 195 200 205 Gln Thr His Cys Cys Ser Gln Asn
Lys Cys Asn Phe Tyr Asp Asn Lys 210 215 220 Asp Leu Glu Cys Val Thr
Asn Leu Gln Glu Val Ala Arg Ile Val Gly 225 230 235 240 Asn Ser Gly
Leu Asn Ile Tyr Asn Leu Tyr Ala Pro Cys Ala Gly Gly 245 250 255 Val
Pro Ser His Phe Arg Tyr Glu Lys Asp Thr Val Val Val Gln Asp 260 265
270 Leu Gly Asn Ile Phe Thr Arg Leu Pro Leu Lys Arg Met Trp His Gln
275 280 285 Ala Leu Leu Arg Ser Gly Asp Lys Val Arg Met Asp Pro Pro
Cys Thr 290 295 300 Asn Thr Thr Ala Ala Ser Thr Tyr Leu Asn Asn Pro
Tyr Val Arg Lys 305 310 315 320 Ala Leu Asn Ile Pro Glu Gln Leu Pro
Gln Trp Asp Met Cys Asn Phe 325 330 335 Leu Val Asn Leu Gln Tyr Arg
Arg Leu Tyr Arg Ser Met Asn Ser Gln 340 345 350 Tyr Leu Lys Leu Leu
Ser Ser Gln Lys Tyr Gln Ile Leu Leu Tyr Asn 355 360 365 Gly Asp Val
Asp Met Ala Cys Asn Phe Met Gly Asp Glu Trp Phe Val 370 375 380 Asp
Ser Leu Asn Gln Lys Met Glu Val Gln Arg Arg Pro Trp Leu Val 385 390
395 400 Lys Tyr Gly Asp Ser Gly Glu Gln Ile Ala Gly Phe Val Lys Glu
Phe 405 410 415
Ser His Ile Ala Phe Leu Thr Ile Lys Gly Ala Gly His Met Val Pro 420
425 430 Thr Asp Lys Pro Leu Ala Ala Phe Thr Met Phe Ser Arg Phe Leu
Asn 435 440 445 Lys Gln Pro Tyr 450 <210> SEQ ID NO 113
<211> LENGTH: 145 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 113 Leu
Pro Gln Ser Phe Leu Leu Lys Cys Leu Glu Gln Val Arg Lys Ile 1 5 10
15 Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys
20 25 30 Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu
Gly Ile 35 40 45 Pro Trp Ala Pro Leu Leu Ala Gly Cys Leu Ser Gln
Leu His Ser Gly 50 55 60 Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala
Leu Glu Gly Ile Ser Pro 65 70 75 80 Glu Leu Gly Pro Thr Leu Asp Thr
Leu Gln Leu Asp Val Ala Asp Phe 85 90 95 Ala Thr Thr Ile Trp Gln
Gln Met Glu Glu Leu Gly Met Met Pro Ala 100 105 110 Phe Ala Ser Ala
Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser 115 120 125 His Leu
Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu 130 135 140
Ala 145 <210> SEQ ID NO 114 <211> LENGTH: 105
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <400> SEQUENCE: 114 Glu His Val Asn Ala Ile Gln
Glu Ala Arg Arg Leu Leu Asn Leu Ser 1 5 10 15 Arg Asp Thr Ala Ala
Glu Met Asn Glu Thr Val Glu Val Ile Ser Glu 20 25 30 Met Phe Asp
Leu Gln Glu Pro Thr Cys Leu Gln Thr Arg Leu Glu Leu 35 40 45 Tyr
Lys Gln Gly Leu Arg Gly Ser Leu Thr Lys Leu Lys Gly Pro Leu 50 55
60 Thr Met Met Ala Ser His Tyr Lys Gln His Cys Pro Pro Thr Pro Glu
65 70 75 80 Thr Ser Cys Ala Thr Gln Ile Ile Thr Phe Glu Ser Phe Lys
Glu Asn 85 90 95 Leu Lys Asp Phe Leu Leu Val Ile Pro 100 105
<210> SEQ ID NO 115 <211> LENGTH: 165 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 115 Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg
Thr Leu Met 1 5 10 15 Leu Leu Ala Gln Met Arg Lys Ile Ser Leu Phe
Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Gly Phe Pro Gln Glu
Glu Phe Gly Asn Gln Phe Gln 35 40 45 Lys Ala Glu Thr Ile Pro Val
Leu His Glu Met Ile Gln Gln Ile Phe 50 55 60 Asn Leu Phe Ser Thr
Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu 65 70 75 80 Leu Asp Lys
Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu 85 90 95 Ala
Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys 100 105
110 Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu
115 120 125 Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val
Val Arg 130 135 140 Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn
Leu Gln Glu Ser 145 150 155 160 Leu Arg Ser Lys Glu 165 <210>
SEQ ID NO 116 <211> LENGTH: 166 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 116 Met Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser Ser
Asn Phe Gln 1 5 10 15 Cys Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg
Leu Glu Tyr Cys Leu 20 25 30 Lys Asp Arg Met Asn Phe Asp Ile Pro
Glu Glu Ile Lys Gln Leu Gln 35 40 45 Gln Phe Gln Lys Glu Asp Ala
Ala Leu Thr Ile Tyr Glu Met Leu Gln 50 55 60 Asn Ile Phe Ala Ile
Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp Asn 65 70 75 80 Glu Thr Ile
Val Glu Asn Leu Leu Ala Asn Val Tyr His Gln Ile Asn 85 90 95 His
Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe Thr 100 105
110 Arg Gly Lys Leu Met Ser Ser Leu His Leu Lys Arg Tyr Tyr Gly Arg
115 120 125 Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala
Trp Thr 130 135 140 Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe
Ile Asn Arg Leu 145 150 155 160 Thr Gly Tyr Leu Arg Asn 165
<210> SEQ ID NO 117 <211> LENGTH: 242 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 117 Met Gln Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys
Lys Tyr Phe 1 5 10 15 Asn Ala Gly His Ser Asp Val Ala Asp Asn Gly
Thr Leu Phe Leu Gly 20 25 30 Ile Leu Lys Asn Trp Lys Glu Glu Ser
Asp Arg Lys Ile Met Gln Ser 35 40 45 Gln Ile Val Ser Phe Tyr Phe
Lys Leu Phe Lys Asn Phe Lys Asp Asp 50 55 60 Gln Ser Ile Gln Lys
Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val 65 70 75 80 Lys Phe Phe
Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu 85 90 95 Thr
Asn Tyr Ser Val Thr Asp Leu Asn Val Gln Arg Lys Ala Ile Asp 100 105
110 Glu Leu Ile Gln Val Met Ala Glu Leu Gly Ala Asn Val Ser Gly Glu
115 120 125 Phe Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr Phe Asn Asp
Asn Gly 130 135 140 Thr Leu Phe Leu Gly Ile Leu Lys Asn Trp Lys Glu
Glu Ser Asp Arg 145 150 155 160 Lys Ile Met Gln Ser Gln Ile Val Ser
Phe Tyr Phe Lys Leu Phe Lys 165 170 175 Asn Phe Lys Asp Asp Gln Ser
Ile Gln Lys Ser Val Glu Thr Ile Lys 180 185 190 Glu Asp Met Asn Val
Lys Phe Phe Asn Ser Asn Lys Lys Lys Arg Asp 195 200 205 Asp Phe Glu
Lys Leu Thr Asn Tyr Ser Val Thr Asp Leu Asn Val Gln 210 215 220 Arg
Lys Ala Ile His Glu Leu Ile Gln Val Met Ala Glu Leu Ser Pro 225 230
235 240 Ala Ala <210> SEQ ID NO 118 <211> LENGTH: 122
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <400> SEQUENCE: 118 Ser Thr Lys Lys Thr Gln Leu
Gln Leu Glu His Leu Leu Leu Asp Leu 1 5 10 15 Gln Met Ile Leu Asn
Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr 20 25 30 Arg Met Leu
Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu 35 40 45 Lys
His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val 50 55
60 Leu Asn Leu Ala Gln Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser
65 70 75 80 Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Phe Met Cys
Glu Tyr 85 90 95
Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr 100
105 110 Phe Cys Gln Ser Ile Ile Ser Thr Leu Thr 115 120 <210>
SEQ ID NO 119 <211> LENGTH: 152 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 119 Ala Pro Val Arg Ser Leu Asn Cys Thr Leu Arg Asp Ser
Gln Gln Lys 1 5 10 15 Ser Leu Val Met Ser Gly Pro Tyr Glu Leu Lys
Ala Leu His Leu Gln 20 25 30 Gly Gln Asp Met Glu Gln Gln Val Val
Phe Ser Met Ser Phe Val Gln 35 40 45 Gly Glu Glu Ser Asn Asp Lys
Ile Pro Val Ala Leu Gly Leu Lys Glu 50 55 60 Lys Asn Leu Tyr Leu
Ser Cys Val Leu Lys Asp Asp Lys Pro Thr Leu 65 70 75 80 Gln Leu Glu
Ser Val Asp Pro Lys Asn Tyr Pro Lys Lys Lys Met Glu 85 90 95 Lys
Arg Phe Val Phe Asn Lys Ile Glu Ile Asn Asn Lys Leu Glu Phe 100 105
110 Glu Ser Ala Gln Phe Pro Asn Trp Tyr Ile Ser Thr Ser Gln Ala Glu
115 120 125 Asn Met Pro Val Phe Leu Gly Gly Thr Lys Gly Gly Gln Asp
Ile Thr 130 135 140 Asp Phe Thr Met Gln Phe Val Ser 145 150
<210> SEQ ID NO 120 <211> LENGTH: 148 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 120 Asp Lys Pro Val Ala His Val Val Ala Asn Pro Gln Ala
Glu Gly Gln 1 5 10 15 Leu Gln Trp Ser Asn Arg Arg Ala Asn Ala Leu
Leu Ala Asn Gly Val 20 25 30 Glu Leu Arg Asp Asn Gln Leu Val Val
Pro Ile Glu Gly Leu Phe Leu 35 40 45 Ile Tyr Ser Gln Val Leu Phe
Lys Gly Gln Gly Cys Pro Ser Thr His 50 55 60 Val Leu Leu Thr His
Thr Ile Ser Arg Ile Ala Val Ser Tyr Gln Thr 65 70 75 80 Lys Val Asn
Leu Leu Ser Ala Ile Lys Ser Pro Cys Gln Arg Glu Thr 85 90 95 Pro
Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu Pro Ile Tyr Leu Gly 100 105
110 Gly Val Phe Gln Leu Glu Lys Gly Asp Arg Leu Ser Ala Glu Ile Asn
115 120 125 Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser Gly Gln Val Tyr
Phe Gly 130 135 140 Ile Ile Ala Leu 145 <210> SEQ ID NO 121
<211> LENGTH: 144 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 121 Lys
Pro Ala Ala His Leu Ile Gly Asp Pro Ser Lys Gln Asn Ser Leu 1 5 10
15 Leu Trp Arg Ala Asn Thr Asp Arg Ala Phe Leu Gln Asp Gly Phe Ser
20 25 30 Leu Ser Asn Asn Ser Leu Leu Val Pro Thr Ser Gly Ile Tyr
Phe Val 35 40 45 Tyr Ser Gln Val Val Phe Ser Gly Lys Ala Tyr Ser
Pro Lys Ala Thr 50 55 60 Ser Ser Pro Leu Tyr Leu Ala His Glu Val
Gln Leu Phe Ser Ser Gln 65 70 75 80 Tyr Pro Phe His Val Pro Leu Leu
Ser Ser Gln Lys Met Val Tyr Pro 85 90 95 Gly Leu Gln Glu Pro Trp
Leu His Ser Met Tyr His Gly Ala Ala Phe 100 105 110 Gln Leu Thr Gln
Gly Asp Gln Leu Ser Thr His Thr Asp Gly Ile Pro 115 120 125 His Leu
Val Leu Ser Pro Ser Thr Val Phe Phe Gly Ala Phe Ala Leu 130 135 140
<210> SEQ ID NO 122 <211> LENGTH: 166 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 122 Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg Val Leu Glu
Arg Tyr Leu 1 5 10 15 Leu Glu Ala Lys Glu Ala Glu Lys Ile Thr Thr
Gly Cys Ala Glu His 20 25 30 Cys Ser Leu Asn Glu Lys Ile Thr Val
Pro Asp Thr Lys Val Asn Phe 35 40 45 Tyr Ala Trp Lys Arg Met Glu
Val Gly Gln Gln Ala Val Glu Val Trp 50 55 60 Gln Gly Leu Ala Leu
Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu 65 70 75 80 Leu Val Lys
Ser Ser Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp 85 90 95 Lys
Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu 100 105
110 Gly Ala Gln Lys Glu Ala Ile Ser Asn Ser Asp Ala Ala Ser Ala Ala
115 120 125 Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe
Arg Val 130 135 140 Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr
Thr Gly Glu Ala 145 150 155 160 Cys Arg Thr Gly Asp Arg 165
<210> SEQ ID NO 123 <211> LENGTH: 21 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 123 Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu
Tyr Gln Leu 1 5 10 15 Glu Asn Tyr Cys Asn 20 <210> SEQ ID NO
124 <211> LENGTH: 29 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 124
Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr 1 5
10 15 Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys 20 25
<210> SEQ ID NO 125 <211> LENGTH: 166 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 125 Phe Pro Thr Ile Pro Leu Ser Arg Leu Ala Asp Asn Ala
Trp Leu Arg 1 5 10 15 Ala Asp Arg Leu Asn Gln Leu Ala Phe Asp Thr
Tyr Gln Glu Phe Glu 20 25 30 Glu Ala Tyr Ile Pro Lys Glu Gln Ile
His Ser Phe Trp Trp Asn Pro 35 40 45 Gln Thr Ser Leu Cys Pro Ser
Glu Ser Ile Pro Thr Pro Ser Asn Lys 50 55 60 Glu Glu Thr Gln Gln
Lys Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu 65 70 75 80 Leu Leu Ile
Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg Ser Val 85 90 95 Phe
Ala Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp 100 105
110 Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly Arg Leu
115 120 125 Glu Ala Leu Leu Lys Asn Tyr Gly Leu Leu Tyr Cys Phe Asn
Lys Asp 130 135 140 Met Ser Lys Val Ser Thr Tyr Leu Arg Thr Val Gln
Cys Arg Ser Val 145 150 155 160 Glu Gly Ser Cys Gly Phe 165
<210> SEQ ID NO 126 <211> LENGTH: 242 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 126
Cys His His Arg Ile Cys His Cys Ser Asn Arg Val Phe Leu Cys Gln 1 5
10 15 Glu Ser Lys Val Thr Glu Ile Pro Ser Asp Leu Pro Arg Asn Ala
Ile 20 25 30 Glu Leu Arg Phe Val Leu Thr Lys Leu Arg Val Ile Gln
Lys Gly Ala 35 40 45 Phe Ser Gly Phe Gly Asp Leu Glu Lys Ile Glu
Ile Ser Gln Asn Asp 50 55 60 Val Leu Glu Val Ile Glu Ala Asp Val
Phe Ser Asn Leu Pro Lys Leu 65 70 75 80 His Glu Ile Arg Ile Glu Lys
Ala Asn Asn Leu Leu Tyr Ile Asn Pro 85 90 95 Glu Ala Phe Gln Asn
Leu Pro Asn Leu Gln Tyr Leu Leu Ile Ser Asn 100 105 110 Thr Gly Ile
Lys His Leu Pro Asp Val His Lys Ile His Ser Leu Gln 115 120 125 Lys
Val Leu Leu Asp Ile Gln Asp Asn Ile Asn Ile His Thr Ile Glu 130 135
140 Arg Asn Ser Phe Val Gly Leu Ser Phe Glu Ser Val Ile Leu Trp Leu
145 150 155 160 Asn Lys Asn Gly Ile Gln Glu Ile His Asn Cys Ala Phe
Asn Gly Thr 165 170 175 Gln Leu Asp Glu Leu Asn Leu Ser Asp Asn Asn
Asn Leu Glu Glu Leu 180 185 190 Pro Asn Asp Val Phe His Gly Ala Ser
Gly Pro Val Ile Leu Asp Ile 195 200 205 Ser Arg Thr Arg Ile His Ser
Leu Pro Ser Tyr Gly Leu Glu Asn Leu 210 215 220 Lys Lys Leu Arg Ala
Arg Ser Thr Tyr Asn Leu Lys Lys Leu Pro Thr 225 230 235 240 Leu Glu
<210> SEQ ID NO 127 <211> LENGTH: 130 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 127 Ile Gln Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys
Thr Ile Val 1 5 10 15 Thr Arg Ile Asn Asp Ile Leu Asp Phe Ile Pro
Gly Leu His Pro Ile 20 25 30 Leu Thr Leu Ser Lys Met Asp Gln Thr
Leu Ala Val Tyr Gln Gln Ile 35 40 45 Leu Thr Ser Met Pro Ser Arg
Asn Val Ile Gln Ile Ser Asn Asp Leu 50 55 60 Glu Asn Leu Arg Asp
Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys 65 70 75 80 His Leu Pro
Glu Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly 85 90 95 Val
Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg 100 105
110 Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln Leu Asp Leu Ser Pro
115 120 125 Gly Cys 130 <210> SEQ ID NO 128 <211>
LENGTH: 62 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Polypeptide <400> SEQUENCE: 128 Pro Glu Thr Leu Cys
Gly Ala Glu Leu Val Asp Ala Leu Gln Phe Val 1 5 10 15 Cys Gly Asp
Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly Ser 20 25 30 Ser
Ser Arg Arg Ala Pro Gln Thr Gly Ile Val Asp Glu Cys Cys Phe 35 40
45 Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys Ala Pro 50 55 60
<210> SEQ ID NO 129 <211> LENGTH: 125 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 129 Met Tyr Arg Ser Ala Phe Ser Val Gly Leu Glu Thr Arg
Val Thr Val 1 5 10 15 Pro Asn Val Pro Ile Arg Phe Thr Lys Ile Phe
Tyr Asn Gln Gln Asn 20 25 30 His Tyr Asp Gly Ser Thr Gly Lys Phe
Tyr Cys Asn Ile Pro Gly Leu 35 40 45 Tyr Tyr Phe Ser Tyr His Ile
Thr Val Tyr Met Lys Asp Val Lys Val 50 55 60 Ser Leu Phe Lys Lys
Asp Lys Ala Val Leu Phe Thr Tyr Asp Gln Tyr 65 70 75 80 Gln Glu Asn
Val Asp Gln Ala Ser Gly Ser Val Leu Leu His Leu Glu 85 90 95 Val
Gly Asp Gln Val Trp Leu Gln Val Tyr Tyr Ala Asp Asn Val Asn 100 105
110 Asp Ser Thr Phe Thr Gly Phe Leu Leu Tyr His Asp Thr 115 120 125
<210> SEQ ID NO 130 <211> LENGTH: 111 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 130 Met Tyr Arg Ser Ala Phe Ser Val Gly Leu Pro Asn Val
Pro Ile Arg 1 5 10 15 Phe Thr Lys Ile Phe Tyr Asn Gln Gln Asn His
Tyr Asp Gly Ser Thr 20 25 30 Gly Lys Phe Tyr Cys Asn Ile Pro Gly
Leu Tyr Tyr Phe Ser Tyr His 35 40 45 Ile Thr Val Tyr Met Lys Asp
Val Lys Val Ser Leu Phe Lys Lys Asp 50 55 60 Lys Val Leu Phe Thr
Tyr Asp Gln Tyr Gln Glu Lys Val Asp Gln Ala 65 70 75 80 Ser Gly Ser
Val Leu Leu His Leu Glu Val Gly Asp Gln Val Trp Leu 85 90 95 Gln
Val Tyr Asp Ser Thr Phe Thr Gly Phe Leu Leu Tyr His Asp 100 105 110
<210> SEQ ID NO 131 <211> LENGTH: 102 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 131 Met Tyr Arg Ser Ala Phe Ser Val Gly Leu Glu Thr Arg
Val Thr Val 1 5 10 15 Pro Ile Arg Phe Thr Lys Ile Phe Tyr Asn Gln
Gln Asn His Tyr Asp 20 25 30 Gly Ser Thr Gly Lys Phe Tyr Cys Asn
Ile Pro Gly Leu Tyr Tyr Phe 35 40 45 Ser Tyr His Ile Thr Val Asp
Val Lys Val Ser Leu Phe Lys Lys Asp 50 55 60 Lys Ala Val Leu Phe
Thr Gln Ala Ser Gly Ser Val Leu Leu His Leu 65 70 75 80 Glu Val Gly
Asp Gln Val Trp Leu Gln Asn Asp Ser Thr Phe Thr Gly 85 90 95 Phe
Leu Leu Tyr His Asp 100 <210> SEQ ID NO 132 <211>
LENGTH: 693 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Polypeptide <400> SEQUENCE: 132 Ala Thr Arg Arg Tyr
Tyr Leu Gly Ala Val Glu Leu Ser Trp Asp Tyr 1 5 10 15 Met Gln Ser
Asp Leu Gly Glu Leu Pro Val Asp Ala Arg Phe Pro Pro 20 25 30 Arg
Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val Tyr Lys Lys 35 40
45 Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile Ala Lys Pro
50 55 60 Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln Ala
Glu Val 65 70 75 80 Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala
Ser His Pro Val 85 90 95 Ser Leu His Ala Val Gly Val Ser Tyr Trp
Lys Ala Ser Glu Gly Ala 100 105 110 Glu Tyr Asp Asp Gln Thr Ser Gln
Arg Glu Lys Glu Asp Asp Lys Val 115 120 125 Phe Pro Gly Gly Ser His
Thr Tyr Val Trp Gln Val Leu Lys Glu Asn 130 135 140 Gly Pro Met Ala
Ser Asp Pro Leu Cys Leu Thr Tyr Ser Tyr Leu Ser 145 150 155 160 His
Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala Leu 165 170
175 Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr Leu
180 185 190 His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly Lys
Ser Trp 195 200 205
His Ser Glu Thr Lys Asn Ala Ala Ser Ala Arg Ala Trp Pro Lys Met 210
215 220 His Thr Val Asn Gly Tyr Val Asn Arg Ser Leu Pro Gly Leu Ile
Gly 225 230 235 240 Cys His Arg Lys Ser Val Tyr Trp His Val Ile Gly
Met Gly Thr Thr 245 250 255 Pro Glu Val His Ser Ile Phe Leu Glu Gly
His Thr Phe Leu Val Arg 260 265 270 Asn His Arg Gln Ala Ser Leu Glu
Ile Ser Pro Ile Thr Phe Leu Thr 275 280 285 Ala Gln Thr Leu Leu Met
Asp Leu Gly Gln Phe Leu Leu Phe Cys His 290 295 300 Ile Ser Ser His
Gln His Asp Gly Met Glu Ala Tyr Val Lys Val Asp 305 310 315 320 Ser
Cys Pro Glu Glu Pro Gln Phe Asp Asp Asp Asn Ser Pro Ser Phe 325 330
335 Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr Trp Val His
340 345 350 Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro Leu
Val Leu 355 360 365 Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu
Asn Asn Gly Pro 370 375 380 Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val
Arg Phe Met Ala Tyr Thr 385 390 395 400 Asp Glu Thr Phe Lys Thr Arg
Glu Ala Ile Gln His Glu Ser Gly Ile 405 410 415 Leu Gly Pro Leu Leu
Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile Ile 420 425 430 Phe Lys Asn
Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile 435 440 445 Thr
Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys Gly Val Lys 450 455
460 His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe Lys Tyr Lys
465 470 475 480 Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp
Pro Arg Cys 485 490 495 Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met
Glu Arg Asp Leu Ala 500 505 510 Ser Gly Leu Ile Gly Pro Leu Leu Ile
Cys Tyr Lys Glu Ser Val Asp 515 520 525 Gln Arg Gly Asn Gln Ile Met
Ser Asp Lys Arg Asn Val Ile Leu Phe 530 535 540 Ser Val Phe Asp Glu
Asn Arg Ser Trp Tyr Leu Thr Glu Asn Ile Gln 545 550 555 560 Arg Phe
Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp Pro Glu Phe 565 570 575
Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val Phe Asp Ser 580
585 590 Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp Tyr Ile
Leu 595 600 605 Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe
Ser Gly Tyr 610 615 620 Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr
Leu Thr Leu Phe Pro 625 630 635 640 Phe Ser Gly Glu Thr Val Phe Met
Ser Met Glu Asn Pro Gly Leu Trp 645 650 655 Ile Leu Gly Cys His Asn
Ser Asp Phe Arg Asn Arg Gly Met Thr Ala 660 665 670 Leu Leu Lys Val
Ser Ser Cys Asp Lys Asn Thr Gly Asp Tyr Tyr Glu 675 680 685 Asp Ser
Tyr Glu Asp 690 <210> SEQ ID NO 133 <211> LENGTH: 644
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <400> SEQUENCE: 133 Arg Ser Phe Gln Lys Lys Thr
Arg His Tyr Phe Ile Ala Ala Val Glu 1 5 10 15 Arg Leu Trp Asp Tyr
Gly Met Ser Ser Ser Pro His Val Leu Arg Asn 20 25 30 Arg Ala Gln
Ser Gly Ser Val Pro Gln Phe Lys Lys Val Val Phe Gln 35 40 45 Glu
Phe Thr Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu 50 55
60 Asn Glu His Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu
65 70 75 80 Asp Asn Ile Met Val Thr Phe Arg Asn Gln Ala Ser Arg Pro
Tyr Ser 85 90 95 Phe Tyr Ser Ser Leu Ile Ser Tyr Glu Glu Asp Gln
Arg Gln Gly Ala 100 105 110 Glu Pro Arg Lys Asn Phe Val Lys Pro Asn
Glu Thr Lys Thr Tyr Phe 115 120 125 Trp Lys Val Gln His His Met Ala
Pro Thr Lys Asp Glu Phe Asp Cys 130 135 140 Lys Ala Trp Ala Tyr Ser
Ser Asp Val Asp Leu Glu Lys Asp Val His 145 150 155 160 Ser Gly Leu
Ile Gly Pro Leu Leu Val Cys His Thr Asn Thr Leu Asn 165 170 175 Pro
Ala His Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu Phe Phe 180 185
190 Thr Ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu Asn Met Glu
195 200 205 Arg Asn Cys Arg Ala Pro Cys Asn Ile Gln Met Glu Asp Pro
Thr Phe 210 215 220 Lys Glu Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr
Ile Met Asp Thr 225 230 235 240 Leu Pro Gly Leu Val Met Ala Gln Asp
Gln Arg Ile Arg Trp Tyr Leu 245 250 255 Leu Ser Met Gly Ser Asn Glu
Asn Ile His Ser Ile His Phe Ser Gly 260 265 270 His Val Phe Thr Val
Arg Lys Lys Glu Glu Tyr Lys Met Ala Leu Tyr 275 280 285 Asn Leu Tyr
Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser Lys 290 295 300 Ala
Gly Ile Trp Arg Val Glu Cys Leu Ile Gly Glu His Leu His Ala 305 310
315 320 Gly Met Ser Thr Leu Phe Leu Val Tyr Ser Asn Lys Cys Gln Thr
Pro 325 330 335 Leu Gly Met Ala Ser Gly His Ile Arg Asp Phe Gln Ile
Thr Ala Ser 340 345 350 Gly Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala
Arg Leu His Tyr Ser 355 360 365 Gly Ser Ile Asn Ala Trp Ser Thr Lys
Glu Pro Phe Ser Trp Ile Lys 370 375 380 Val Asp Leu Leu Ala Pro Met
Ile Ile His Gly Ile Lys Thr Gln Gly 385 390 395 400 Ala Arg Gln Lys
Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile Met 405 410 415 Tyr Ser
Leu Asp Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn Ser Thr 420 425 430
Gly Thr Leu Met Val Phe Phe Gly Asn Val Asp Ser Ser Gly Ile Lys 435
440 445 His Asn Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr Ile Arg Leu
His 450 455 460 Pro Thr His Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu
Leu Met Gly 465 470 475 480 Cys Asp Leu Asn Ser Cys Ser Met Pro Leu
Gly Met Glu Ser Lys Ala 485 490 495 Ile Ser Asp Ala Gln Ile Thr Ala
Ser Ser Tyr Phe Thr Asn Met Phe 500 505 510 Ala Thr Trp Ser Pro Ser
Lys Ala Arg Leu His Leu Gln Gly Arg Ser 515 520 525 Asn Ala Trp Arg
Pro Gln Val Asn Asn Pro Lys Glu Trp Leu Gln Val 530 535 540 Asp Phe
Gln Lys Thr Met Lys Val Thr Gly Val Thr Thr Gln Gly Val 545 550 555
560 Lys Ser Leu Leu Thr Ser Met Tyr Val Lys Glu Phe Leu Ile Ser Ser
565 570 575 Ser Gln Asp Gly His Gln Trp Thr Leu Phe Phe Gln Asn Gly
Lys Val 580 585 590 Lys Val Phe Gln Gly Asn Gln Asp Ser Phe Thr Pro
Val Val Asn Ser 595 600 605 Leu Asp Pro Pro Leu Leu Thr Arg Tyr Leu
Arg Ile His Pro Gln Ser 610 615 620 Trp Val His Gln Ile Ala Leu Arg
Met Glu Val Leu Gly Cys Glu Ala 625 630 635 640 Gln Asp Leu Tyr
<210> SEQ ID NO 134 <211> LENGTH: 578 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 134 Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu Glu
Asn Phe Lys 1 5 10 15 Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu
Gln Gln Cys Pro Phe 20 25 30 Glu Asp His Val Lys Leu Val Asn Glu
Val Thr Glu Phe Ala Lys Thr 35 40 45 Cys Val Ala Asp Glu Ser Ala
Glu Asn Cys Asp Lys Ser Leu His Thr 50 55 60 Leu Phe Gly Asp Lys
Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr 65 70 75 80 Gly Glu Met
Ala Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu
85 90 95 Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu Pro Arg
Leu Val 100 105 110 Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His
Asp Asn Glu Glu 115 120 125 Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile
Ala Arg Arg His Pro Tyr 130 135 140 Phe Tyr Ala Pro Glu Leu Leu Phe
Phe Ala Lys Arg Tyr Lys Ala Ala 145 150 155 160 Phe Thr Glu Cys Cys
Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro 165 170 175 Lys Leu Asp
Glu Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln 180 185 190 Arg
Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys 195 200
205 Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe
210 215 220 Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys Val His
Thr Glu 225 230 235 240 Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp
Asp Arg Ala Asp Leu 245 250 255 Ala Lys Tyr Ile Cys Glu Asn Gln Asp
Ser Ile Ser Ser Lys Leu Lys 260 265 270 Glu Cys Cys Glu Lys Pro Leu
Leu Glu Lys Ser His Cys Ile Ala Glu 275 280 285 Val Glu Asn Asp Glu
Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp 290 295 300 Phe Val Glu
Ser Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp 305 310 315 320
Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp 325
330 335 Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr
Thr 340 345 350 Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu Cys
Tyr Ala Lys 355 360 365 Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu
Pro Gln Asn Leu Ile 370 375 380 Lys Gln Asn Cys Glu Leu Phe Glu Gln
Leu Gly Glu Tyr Lys Phe Gln 385 390 395 400 Asn Ala Leu Leu Val Arg
Tyr Thr Lys Lys Val Pro Gln Val Ser Thr 405 410 415 Pro Thr Leu Val
Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys 420 425 430 Cys Cys
Lys His Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr 435 440 445
Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro 450
455 460 Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn
Arg 465 470 475 480 Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr
Tyr Val Pro Lys 485 490 495 Glu Phe Asn Ala Glu Thr Phe Thr Phe His
Ala Asp Ile Cys Thr Leu 500 505 510 Ser Glu Lys Glu Arg Gln Ile Lys
Lys Gln Thr Ala Leu Val Glu Leu 515 520 525 Val Lys His Lys Pro Lys
Ala Thr Lys Glu Gln Leu Lys Ala Val Met 530 535 540 Asp Asp Phe Ala
Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys 545 550 555 560 Glu
Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln 565 570
575 Ala Ala <210> SEQ ID NO 135 <211> LENGTH: 578
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <400> SEQUENCE: 135 Ser Glu Val Ala His Arg Phe
Lys Asp Leu Gly Glu Glu Asn Phe Lys 1 5 10 15 Ala Leu Val Leu Ile
Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe 20 25 30 Glu Asp His
Val Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr 35 40 45 Cys
Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr 50 55
60 Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr
65 70 75 80 Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg
Asn Glu 85 90 95 Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu
Pro Arg Leu Val 100 105 110 Arg Pro Glu Val Asp Val Met Cys Thr Ala
Phe His Asp Asn Glu Glu 115 120 125 Thr Phe Leu Lys Lys Tyr Leu Tyr
Glu Ile Ala Arg Arg His Pro Tyr 130 135 140 Phe Tyr Ala Pro Glu Leu
Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala 145 150 155 160 Phe Thr Glu
Cys Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro 165 170 175 Lys
Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln 180 185
190 Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys
195 200 205 Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala
Glu Phe 210 215 220 Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys
Val His Thr Glu 225 230 235 240 Cys Cys His Gly Asp Leu Leu Glu Cys
Ala Asp Asp Arg Ala Asp Leu 245 250 255 Ala Lys Tyr Ile Cys Glu Asn
Gln Asp Ser Ile Ser Ser Lys Leu Lys 260 265 270 Glu Cys Cys Glu Lys
Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu 275 280 285 Val Glu Asn
Asp Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp 290 295 300 Phe
Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp 305 310
315 320 Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro
Asp 325 330 335 Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr Tyr
Glu Thr Thr 340 345 350 Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His
Glu Cys Tyr Ala Lys 355 360 365 Val Phe Asp Glu Phe Lys Pro Leu Val
Glu Glu Pro Gln Asn Leu Ile 370 375 380 Lys Gln Asn Cys Glu Leu Phe
Glu Gln Leu Gly Glu Tyr Lys Phe Gln 385 390 395 400 Asn Ala Leu Leu
Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr 405 410 415 Pro Thr
Leu Val Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys 420 425 430
Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr 435
440 445 Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr
Pro 450 455 460 Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser Leu
Val Asn Arg 465 470 475 480 Arg Pro Cys Phe Ser Ala Leu Glu Val Asp
Glu Thr Tyr Val Pro Lys 485 490 495 Glu Phe Asn Ala Glu Thr Phe Thr
Phe His Ala Asp Ile Cys Thr Leu 500 505 510 Ser Glu Lys Glu Arg Gln
Ile Lys Lys Gln Thr Ala Leu Val Glu Leu 515 520 525 Val Lys His Lys
Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met 530 535 540 Asp Asp
Phe Ala Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys 545 550 555
560 Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln
565 570 575 Ala Ala <210> SEQ ID NO 136 <211> LENGTH:
141 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Polypeptide <400> SEQUENCE: 136 Val Leu Ser Pro Ala Asp Lys
Thr Asn Val Lys Ala Ala Trp Gly Lys 1 5 10 15 Val Gly Ala His Ala
Gly Glu Tyr Gly Ala Glu Ala Leu Glu Arg Met 20 25 30 Phe Leu Ser
Phe Pro Thr Thr Lys Thr Tyr Phe Pro His Phe Asp Leu 35 40 45 Ser
His Gly Ser Ala Gln Val Lys Gly His Gly Lys Lys Val Ala Asp 50 55
60 Ala Leu Thr Asn Ala Val Ala His Val Asp Asp Met Pro Asn Ala Leu
65 70 75 80 Ser Ala Leu Ser Asp Leu His Ala His Lys Leu Arg Val Asp
Pro Val 85 90 95 Asn Phe Lys Leu Leu Ser His Cys Leu Leu Val Thr
Leu Ala Ala His 100 105 110 Leu Pro Ala Glu Phe Thr Pro Ala Val His
Ala Ser Leu Asp Lys Phe 115 120 125 Leu Ala Ser Val Ser Thr Val Leu
Thr Ser Lys Tyr Arg 130 135 140
<210> SEQ ID NO 137 <211> LENGTH: 146 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Polypeptide <400>
SEQUENCE: 137 Val His Leu Thr Pro Glu Glu Lys Ser Ala Val Thr Ala
Leu Trp Gly 1 5 10 15 Lys Val Asn Val Asp Glu Val Gly Gly Glu Ala
Leu Gly Arg Leu Leu 20 25 30 Val Val Tyr Pro Trp Thr Gln Arg Phe
Phe Glu Ser Phe Gly Asp Leu 35 40 45 Ser Thr Pro Asp Ala Val Met
Gly Asn Pro Lys Val Lys Ala His Gly 50 55 60 Lys Lys Val Leu Gly
Ala Phe Ser Asp Gly Leu Ala His Leu Asp Asn 65 70 75 80 Leu Lys Gly
Thr Phe Ala Thr Leu Ser Glu Leu His Cys Asp Lys Leu 85 90 95 His
Val Asp Pro Glu Asn Phe Arg Leu Leu Gly Asn Val Leu Val Cys 100 105
110 Val Leu Ala His His Phe Gly Lys Glu Phe Thr Pro Pro Val Gln Ala
115 120 125 Ala Tyr Gln Lys Val Val Ala Gly Val Ala Asn Ala Leu Ala
His Lys 130 135 140 Tyr His 145 <210> SEQ ID NO 138
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Polypeptide <400> SEQUENCE: 138 His
Ala Leu Pro Glu Thr Gly 1 5
* * * * *
References